WO2023220087A1 - Compositions d'oligonucléotides et procédés associés - Google Patents

Compositions d'oligonucléotides et procédés associés Download PDF

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WO2023220087A1
WO2023220087A1 PCT/US2023/021586 US2023021586W WO2023220087A1 WO 2023220087 A1 WO2023220087 A1 WO 2023220087A1 US 2023021586 W US2023021586 W US 2023021586W WO 2023220087 A1 WO2023220087 A1 WO 2023220087A1
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oligonucleotide
sugar
composition
ime
i2moerg
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PCT/US2023/021586
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WO2023220087A8 (fr
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Joshua Barry COHEN
Justin Bernard KLEE
Duncan Brown
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Amylyx Pharmaceuticals, Inc.
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Publication of WO2023220087A1 publication Critical patent/WO2023220087A1/fr
Publication of WO2023220087A8 publication Critical patent/WO2023220087A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • Oligonucleotides are useful in various applications, e.g., therapeutic, diagnostic, and/or research applications. For example, oligonucleotides targeting various genes can be useful for treatment of conditions, disorders or diseases related to such target genes.
  • the present disclosure provides technologies (e.g., oligonucleotides, compositions, methods, etc.) for treating various conditions, disorders or diseases associated with calpain-2.
  • the present disclosure provides oligonucleotides that comprise various modifications, e.g., nucleobase modifications, sugar modifications, internucleotidic linkage modifications, etc. and can hybridize to a calpain-2 transcript.
  • the present disclosure provides oligonucleotides and compositions thereof that when administered or delivered to a system comprising or expressing a calpain- 2 transcript can reduce the level of a calpain-2 transcript.
  • a provided technology reduces levels of a calpain-2 transcript and/or polypeptide in a system.
  • the present disclosure provides technologies for preventing and/or treating various conditions, disorders or diseases associated with calpain-2.
  • the present disclosure encompasses the recognition that oligonucleotides of certain base sequence may be more effective in reducing levels of calpain-2 transcripts (e.g., calpain-2 mRNA) and/or products thereof (e.g., calpain-2 polypeptides).
  • a base sequence of an oligonucleotide is comprises about 5 or more (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) contiguous nucleobases of ATCAGTTTCTGTAGGCTTCC, GGCATACTGGTTCAGTTGAT, GCTCAGGTCAGGCAGTGGTT, GAGAGCCTTTTTGCAGAGCT, TCCAGCTCTGTGCCTCTAGT, GTTCCAGCTTGGGCAGTTGT or GGAAGCTTAGTCCTTGGCTG, wherein each T is optionally and independently replaced with U.
  • a base sequence of an oligonucleotide is ATCAGTTTCTGTAGGCTTCC, GGCATACTGGTTCAGTTGAT, GCTCAGGTCAGGCAGTGGTT, GAGAGCCTTTTTGCAGAGCT, TCCAGCTCTGTGCCTCTAGT, GTTCCAGCTTGGGCAGTTGT or GGAAGCTTAGTCCTTGGCTG.
  • provided oligonucleotides comprising various modifications, such as nucleobase modifications, sugar modifications, internucleotidic linkage modifications, etc. Various useful modifications are available in the art and may be utilized in accordance with the present disclosure.
  • modifications provide various benefits, e.g., improved stability, binding affinity, pharmacokinetic profiles, pharmacodynamic profiles, etc.
  • provided oligonucleotides comprise various sugar modifications.
  • a modified sugar is a natural RNA sugar with a 2’-OR s modification, wherein R s is an optionally substituted C 1-6 aliphatic, and ⁇ OR s replace the 2’-OH group (a “2’-OR s modified sugar”).
  • R s is optionally substituted C 1-6 alkyl.
  • R s is ⁇ CH 3 .
  • R s is ⁇ CH 2 CH 2 OCH 3 .
  • provided oligonucleotides comprises or consists of a wing-core-wing structure, wherein there are independently about 1-10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleosides in each wing, there are about 5 or more (e.g., about 5-20, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) nucleosides in a core, and each wing independently comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified sugars.
  • each sugar in a wing is independently a modified sugar.
  • each sugar in a wing is independently a 2’-OR s modified sugar.
  • a modified sugar is a 2’-MOE modified sugar (a 2’-OR s modified sugar wherein R s is ⁇ CH 2 CH 2 OCH 3 ).
  • each wing independently comprises one or more (e.g., about 1, 2, 3, 4, 5 or more) 2’-MOE modified sugar.
  • each sugar in a wing is independently a 2’- MOE modified sugar.
  • core regions comprise fewer modified sugars, and/or lower levels of modified sugars, compared to one or both wings. In some embodiments, there are no modified sugars in a core region.
  • each sugar in a core region is independently a nature DNA sugar.
  • provided oligonucleotides comprise modified internucleotidic linkages.
  • modified internucleotidic linkages provide improved properties and/or activities compared to natural phosphate linkages.
  • Various internucleotidic linkages are available in the art and can be utilized in accordance with the present disclosure.
  • a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage ( ⁇ O ⁇ P(O)(SH) ⁇ O ⁇ , which may exist in various salt forms).
  • each linkages in a provided oligonucleotide is a phosphorothioate internucleotidic linkage.
  • the present disclosure provides technologies for preparing oligonucleotides and compositions thereof.
  • provided oligonucleotides and compositions thereof are of high purity.
  • oligonucleotides are provided as diastereomeric mixtures with respect to chiral linkage phosphorus, e.g., in phosphorothioate internucleotidic linkages.
  • one or more diastereomers with respect to chiral linkage phosphorus are enriched in provided compositions.
  • compositions comprising one or more forms of oligonucleotides, e.g., acid forms (e.g., in which natural phosphate linkages exist as –O(P(O)(OH) ⁇ O ⁇ , phosphorothioate internucleotidic linkages exist as –O(P(O)(SH) ⁇ O ⁇ )), salt forms (e.g., in which one or more or all natural phosphate linkages independently exist as salt forms (e.g., sodium salt (–O(P(O)(O ⁇ Na + ) ⁇ O ⁇ ), one or more or all phosphorothioate internucleotidic linkages exist as salt forms (e.g., sodium salt (–O(P(O)(S ⁇ Na + ) ⁇ O ⁇ ), etc.), hydrates, etc.
  • acid forms e.g., in which natural phosphate linkages exist as –O(P(O)(OH) ⁇ O ⁇ , phosphorothioate internucleot
  • oligonucleotides can exist in various salt forms, including pharmaceutically acceptable salts, and in solutions (e.g., various aqueous buffering system), cations may dissociate from anions.
  • the present disclosure provides a pharmaceutical composition comprising a provided oligonucleotide and/or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is or comprises a buffer.
  • a pharmaceutically acceptable carrier is a buffered saline.
  • a pharmaceutically acceptable carrier is artificial cerebrospinal fluid (aCSF).
  • a pharmaceutically acceptable carrier is cerebrospinal fluid.
  • the present disclosure describes useful technologies for assessing oligonucleotide and compositions thereof. Certain useful technologies are described in the Examples. [0012] Provided technologies can be utilized for various purposes. For example, in some embodiments, provided technologies are useful for preventing and/or treating various conditions, disorders or diseases associated with calpain-2. In some embodiments, the present disclosure provides a method for preventing a condition, disorder or disease, comprising administering or delivering to a subject susceptible thereto an effective amount of a provided oligonucleotide. In some embodiments, the present disclosure provides a method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of a provided oligonucleotide.
  • an oligonucleotide is administered or delivered in a pharmaceutical composition.
  • an oligonucleotide is administered or delivered in one or more forms, e.g., in some embodiments, in one or more pharmaceutically acceptable salt forms.
  • an oligonucleotide is administered or delivered in a solution, e.g., in an aCSF solution.
  • aCSF solution e.g., in an aCSF solution.
  • a condition, disorder or disease is a neurodegenerative condition, disorder or disease.
  • a condition, disorder or disease is or comprises Wallerian degeneration.
  • a condition, disorder or disease is associated with Wallerian degeneration.
  • a condition, disorder or disease is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • a condition, disorder or disease is neuropathy.
  • a condition, disorder or disease is peripheral neuropathy.
  • a condition, disorder or disease is peripheral neuropathy induced by chemotherapy.
  • a condition, disorder or disease is Parkinson’s disease.
  • a condition, disorder or disease is Huntington’s disease.
  • a condition, disorder or disease is Alzheimer’s disease. In some embodiments, a condition, disorder or disease is frontotemporal dementia. In some embodiments, a condition, disorder or disease is brain injury. In some embodiments, a condition, disorder or disease is traumatic brain injury. In some embodiments, a condition, disorder or disease is progressive supranuclear palsy. In some embodiments, a condition, disorder or disease is corticobasal degeneration. In some embodiments, a condition, disorder or disease is Wolfram Syndrome. In some embodiments, a condition, disorder or disease is Friedreich's Ataxia. In some embodiments, a condition, disorder or disease is Multiple System Atrophy.
  • a condition, disorder or disease is Spinal Cerebellar Ataxia. In some embodiments, a condition, disorder or disease is Spinal Muscular Atrophy (SMA). In some embodiments, a condition, disorder or disease is Pick's Disease. In some embodiments, a condition, disorder or disease is progressive motor atrophy.
  • SMA Spinal Muscular Atrophy
  • a condition, disorder or disease is Pick's Disease. In some embodiments, a condition, disorder or disease is progressive motor atrophy.
  • the X- axis denotes oligonucleotide; the Y-axis depicts fold-change in CAPN2 expression (top panel), CAPN2 Cp value (middle panel), or RPLP0 Cp value (bottom panel). Dotted horizontal line indicates corresponding fold- change or Cp value from treatment with vehicle-only negative control. Error bars represent standard deviation.
  • Average fold-change of CAPN2 or NEAT1 expression of biological replicates black dots
  • average CAPN2 or NEAT1 Cp value of technical replicates black dots
  • average RPLP0 Cp value of technical replicates black dots
  • the X-axis denotes oligonucleotide
  • the Y-axis depicts fold-change in CAPN2 or NEAT1 expression (top panel), CAPN2 or NEAT1 Cp value (middle panel), or RPLP0 Cp value (bottom panel).
  • Dotted horizontal line indicates corresponding fold-change or Cp value from treatment with vehicle-only negative control. Error bars represent standard deviation.
  • NEAT1 from left to right, vehicle, positive control, and negative control.
  • Cp2 calpain-2
  • Figure 3 Various provided oligonucleotides display no obvious cytotoxicity as compared to vehicle treatment. Various oligonucleotides were confirmed to not significantly change percentage of live cells. Human iPSC-derived glutamatergic neurons were treated with various oligonucleotides by gymnotic uptake for 48 hours.
  • oligonucleotides can reduce levels of calpain-2 (CAPN2) mRNA.
  • CAPN2 calpain-2
  • Provided oligonucleotides were demonstrated to knockdown CAPN2 in a dose-dependent manner.
  • Human iPSC- derived spinal motor neurons were treated with indicated oligonucleotides targeting CAPN2 or negative control (targeting TUG1) at various concentrations (e.g., 20, 6.329, 2.003, 0.634, 0.201, 0.063, 0.020, 0.006 ⁇ M) by gymnotic uptake for 72 hours. Cells were lysed and RNA collected. RNA was used in real time RT- qPCR to quantify CAPN2 knockdown.
  • the X-axis denotes concentration of oligonucleotide; the Y-axis denotes % knockdown of calpain-2 (CAPN2).
  • Each data point represents mean of replicate treatments, except the data points for oligonucleotide 39 at 6.329 ⁇ M and oligonucleotides 14 and 39 at 0.201 ⁇ M which represent a single replicate. Error bars represent standard deviation.
  • Figure 5 Provided oligonucleotides can provide neuroprotective effects. Provided oligonucleotides were demonstrated to reduce NF-L excretion following exposure to toxicity triggers (e.g., vincristine, rotenone, or colchicine).
  • toxicity triggers e.g., vincristine, rotenone, or colchicine
  • Human iPSC-derived spinal motor neurons were treated with indicated oligonucleotide targeting CAPN2 or oligonucleotide targeting TUG1 at 20 ⁇ M by gymnotic uptake for 48 hours.
  • neurons were treated again with indicated oligonucleotides at 20 ⁇ M by gymnotic uptake and various toxicity triggers (e.g., vincristine (at 1.5 nM, 3 nM, or 6 nM), rotenone (at 5 ⁇ M, 10 ⁇ M, or 15 ⁇ M), colchicine (at 10 nM, 100 nM, or 1000 nM)) for 24 hours.
  • vincristine at 1.5 nM, 3 nM, or 6 nM
  • rotenone at 5 ⁇ M, 10 ⁇ M, or 15 ⁇ M
  • colchicine at 10 nM, 100 nM, or 1000 nM
  • the X-axis denotes treatment (e.g., vehicle (TE buffer), negative control oligonucleotide targeting TUG1, oligonucleotide composition 14); the Y-axis depicts relative concentration of NF-L levels following exposure to toxic triggers (e.g., vincristine (A), rotenone (B), colchicine (C)) normalized to vehicle control.
  • toxic triggers e.g., vincristine (A), rotenone (B), colchicine (C)
  • Statistical analysis was performed using one-way ANOVA with Dunnett’s (vehicle vs.
  • oligonucleotides can provide knockdown of calpain-2 (CAPN2) mRNA.
  • CAPN2 calpain-2
  • Human iPSC-derived motor neurons were treated with various oligonucleotide compositions targeting CAPN2 or TUG1 (negative control) by gymnotic uptake for 48 hours. After 48 hours, media was refreshed to remove oligonucleotides. Cells were lysed at various time points (0, 3, 7, 10, 14, 21 days) following removal of oligonucleotides and RNA collected.
  • the X-axis denotes days (D0, D3, D7, D10, D14, D21) after removal of oligonucleotides; the Y- axis depicts % knockdown of CAPN2. Error bars represent standard deviation. [0020] Figure 7. Provided oligonucleotides can provide knockdown of calpain-2 protein.
  • the X-axis denotes days (D0, D3, D7, D10, D14, D21) after removal of oligonucleotides; the Y-axis depicts relative knockdown of calpain-2 protein (% over mean negative control calpain-2 protein level at corresponding time point). Error bars represent standard deviation.
  • Figure 8. Provided oligonucleotides display no significant effect on cell morphology as compared to vehicle treatment. Human iPSC-derived motor neurons were treated with oligonucleotide compositions targeting calpain-2 (CAPN2) or TUG1 (negative control) by gymnotic uptake for 48 hours. After 48 hours, media was refreshed to remove oligonucleotides.
  • CAN2 calpain-2
  • TUG1 negative control
  • Time point (D0, D3, D21) for each row of images is indicated by labels at left; treatment (vehicle (TE buffer), negative control oligonucleotide targeting TUG1, oligonucleotide composition 14, oligonucleotide composition 39) for each column of images is indicated by labels at top.
  • treatment vehicle (TE buffer), negative control oligonucleotide targeting TUG1, oligonucleotide composition 14, oligonucleotide composition 39
  • DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0022] Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments. Definitions [0023] As used herein, the following definitions shall apply unless otherwise indicated.
  • the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional/second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.
  • oligonucleotides and elements thereof e.g., base sequence, sugar modifications, internucleotidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.
  • description of oligonucleotides and elements thereof is from 5’ to 3’.
  • oligonucleotides may be provided and/or utilized as various forms, e.g., salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts.
  • individual oligonucleotides within a composition may be considered to be of the same constitution and/or structure even though, within such composition (e.g., a liquid composition), particular such oligonucleotides might be in different forms, e.g., salt form(s) (and may be dissolved and the oligonucleotide chain may exist as an anion form when, e.g., in a liquid composition) at a particular moment in time.
  • individual internucleotidic linkages along an oligonucleotide chain may be in an acid (H) form, or in one of a plurality of possible salt forms (e.g., a sodium salt, or a salt of a different cation, depending on which ions might be present in the preparation or composition), and will understand that, so long as their acid forms (e.g., replacing all cations, if any, with H + ) are of the same constitution and/or structure, such individual oligonucleotides may properly be considered to be of the same constitution and/or structure.
  • H acid
  • Aliphatic means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or combinations thereof.
  • aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms.
  • aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • Alkyl As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • alkyl has 1-100 carbon atoms.
  • a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C 1 -C 20 for straight chain, C 2 -C 20 for branched chain), and alternatively, about 1-10.
  • cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure.
  • an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C 1 -C 4 for straight chain lower alkyls).
  • Animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and/or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and/or worms.
  • an animal may be a transgenic animal, a genetically-engineered animal and/or a clone.
  • Characteristic portion refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance.
  • a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity.
  • a characteristic portion shares at least one functional characteristic with the intact substance.
  • a “characteristic portion” of a nucleic acid is one that contains a number of, in some embodiments, a continuous stretch of, nucleobases that are characteristic of that nucleic acid.
  • Comparable is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed.
  • comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • Heteroatom means an atom that is not carbon or hydrogen.
  • a heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen (e.g., quaternized forms, forms as in iminium groups, etc.), phosphorus, sulfur, oxygen; etc.).
  • a heteroatom is silicon, phosphorus, oxygen, sulfur or nitrogen. In some embodiments, a heteroatom is silicon, oxygen, sulfur or nitrogen. In some embodiments, a heteroatom is oxygen, sulfur or nitrogen. [0032] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Internucleotidic linkage refers generally to a linkage linking nucleoside units of an oligonucleotide or a nucleic acid.
  • an internucleotidic linkage is a modified internucleotidic linkage (not a natural phosphate linkage).
  • an internucleotidic linkage is a “modified internucleotidic linkage” wherein at least one oxygen atom or ⁇ OH of a phosphodiester linkage is replaced by a different organic or inorganic moiety.
  • a modified internucleotidic linkage is a phosphorothioate linkage.
  • an internucleotidic linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. It is understood by a person of ordinary skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and/or microbe).
  • in vivo refers to events that occur within an organism (e.g., animal, plant and/or microbe).
  • Linkage phosphorus as defined herein, the phrase “linkage phosphorus” is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotidic linkage as occurs in naturally occurring DNA and RNA.
  • a linkage phosphorus atom is in a modified internucleotidic linkage, wherein each oxygen atom of a phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety.
  • a linkage phosphorus atom is chiral (e.g., as in phosphorothioate internucleotidic linkages). In some embodiments, a linkage phosphorus atom is achiral (e.g., as in natural phosphate linkages).
  • Modified nucleobase The terms "modified nucleobase”, “modified base” and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase which comprises a modification.
  • a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.
  • a modified nucleobase is substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U.
  • a modified nucleobase in the context of oligonucleotides refer to a nucleobase that is not A, T, C, G or U.
  • Modified nucleoside refers to a moiety derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside.
  • modified nucleosides include those which comprise a modification at the base and/or the sugar.
  • modified nucleosides include those with a 2’ modification at a sugar.
  • modified nucleosides also include abasic nucleosides (which lack a nucleobase).
  • a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.
  • Modified nucleotide includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide.
  • a modified nucleotide comprises a modification at a sugar, base and/or internucleotidic linkage.
  • a modified nucleotide comprises a modified sugar, modified nucleobase and/or modified internucleotidic linkage.
  • a modified nucleotide is capable of at least one function of a nucleotide, e.g., forming a subunit in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.
  • Modified sugar refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar.
  • a modified sugar is substituted ribose or deoxyribose.
  • a modified sugar comprises a 2’-modification. Examples of useful 2’- modification are widely utilized in the art and described herein.
  • a 2’-modification is 2’-F.
  • a 2’-modification is 2’-OR, wherein R is optionally substituted C 1-10 aliphatic.
  • a 2’-modification is 2’-OMe.
  • a 2’-modification is 2’-MOE.
  • a modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.).
  • a modified sugar in the context of oligonucleotides, is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA.
  • Nucleic acid includes any nucleotides and polymers thereof.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof.
  • RNA or DNA comprising modified nucleotides and/or modified polynucleotides, such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides.
  • RNA poly- or oligo-ribonucleotides
  • DNA poly- or oligo-deoxyribonucleotides
  • RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases
  • nucleic acids derived from sugars and/or modified sugars and nucleic acids derived from phosphate bridges and/or modified internucleotidic linkages.
  • the term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages.
  • nucleic acids containing ribose moieties examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties.
  • the prefix poly- refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.
  • Nucleobase refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner.
  • the most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).
  • a naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine.
  • a naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine.
  • a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety.
  • a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety.
  • a nucleobase is a “modified nucleobase,” a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).
  • a modified nucleobase is substituted A, T, C, G or U.
  • a modified nucleobase is a substituted tautomer of A, T, C, G, or U.
  • a modified nucleobases is methylated adenine, guanine, uracil, cytosine, or thymine.
  • a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner.
  • a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex.
  • nucleobase also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs.
  • a nucleobase is optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.
  • a “nucleobase” refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).
  • nucleoside refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar.
  • a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine.
  • a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine.
  • a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine.
  • nucleoside refers to a nucleoside unit in an oligonucleotide or a nucleic acid.
  • Nucleotide refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more internucleotidic linkages (e.g., phosphate linkages in natural DNA and RNA).
  • the naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included.
  • the naturally occurring sugar is the pentose (five- carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides.
  • a natural nucleotide comprises a naturally occurring base, sugar and internucleotidic linkage.
  • nucleotide also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleotides and nucleotide analogs.
  • a “nucleotide” refers to a nucleotide unit in an oligonucleotide or a nucleic acid.
  • Oligonucleotide refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and non-natural nucleobases, sugars, and internucleotidic linkages.
  • Oligonucleotides can be single-stranded or double-stranded.
  • a single-stranded oligonucleotide can have double-stranded regions (formed by two portions of the single-stranded oligonucleotide) and a double-stranded oligonucleotide, which comprises two oligonucleotide chains, can have single-stranded regions for example, at regions where the two oligonucleotide chains are not complementary to each other.
  • Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double- stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immuno- stimulatory oligonucleotides, and decoy oligonucleotides.
  • RNAi agents or iRNA agents RNA interference reagents
  • shRNA antisense oligonucleotides
  • ribozymes microRNAs
  • microRNA mimics supermirs
  • aptamers antimirs
  • Oligonucleotides of the present disclosure can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, or triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, an oligonucleotide is from about 9 to about 39 nucleosides in length.
  • an oligonucleotide is from about 25 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 26 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 27 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 28 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 29 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 30 to about 70 nucleosides in length.
  • an oligonucleotide is from about 31 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 32 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 60 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 50 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 40 nucleosides in length. In some embodiments, an oligonucleotide is from about 30 to about 40 nucleosides in length.
  • the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 4 nucleosides in length. In some embodiments, an oligonucleotide is at least 5 nucleosides in length. In some embodiments, an oligonucleotide is at least 6 nucleosides in length. In some embodiments, an oligonucleotide is at least 7 nucleosides in length. In some embodiments, an oligonucleotide is at least 8 nucleosides in length.
  • an oligonucleotide is at least 9 nucleosides in length. In some embodiments, an oligonucleotide is at least 10 nucleosides in length. In some embodiments, an oligonucleotide is at least 11 nucleosides in length. In some embodiments, an oligonucleotide is at least 12 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 16 nucleosides in length.
  • an oligonucleotide is at least 17 nucleosides in length. In some embodiments, an oligonucleotide is at least 18 nucleosides in length. In some embodiments, an oligonucleotide is at least 19 nucleosides in length. In some embodiments, an oligonucleotide is at least 20 nucleosides in length. In some embodiments, an oligonucleotide is at least 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 26 nucleosides in length. In some embodiments, an oligonucleotide is at least 27 nucleosides in length.
  • an oligonucleotide is at least 28 nucleosides in length. In some embodiments, an oligonucleotide is at least 29 nucleosides in length. In some embodiments, an oligonucleotide is at least 30 nucleosides in length. In some embodiments, an oligonucleotide is at least 31 nucleosides in length. In some embodiments, an oligonucleotide is at least 32 nucleosides in length. In some embodiments, an oligonucleotide is at least 33 nucleosides in length. In some embodiments, an oligonucleotide is at least 34 nucleosides in length.
  • an oligonucleotide is at least 35 nucleosides in length. In some embodiments, an oligonucleotide is at least 36 nucleosides in length. In some embodiments, an oligonucleotide is at least 37 nucleosides in length. In some embodiments, an oligonucleotide is at least 38 nucleosides in length. In some embodiments, an oligonucleotide is at least 39 nucleosides in length. In some embodiments, an oligonucleotide is at least 40 nucleosides in length. In some embodiments, an oligonucleotide is 25 nucleosides in length.
  • an oligonucleotide is 26 nucleosides in length. In some embodiments, an oligonucleotide is 27 nucleosides in length. In some embodiments, an oligonucleotide is 28 nucleosides in length. In some embodiments, an oligonucleotide is 29 nucleosides in length. In some embodiments, an oligonucleotide is 30 nucleosides in length. In some embodiments, an oligonucleotide is 31 nucleosides in length. In some embodiments, an oligonucleotide is 32 nucleosides in length. In some embodiments, an oligonucleotide is 33 nucleosides in length.
  • an oligonucleotide is 34 nucleosides in length. In some embodiments, an oligonucleotide is 35 nucleosides in length. In some embodiments, an oligonucleotide is 36 nucleosides in length. In some embodiments, an oligonucleotide is 37 nucleosides in length. In some embodiments, an oligonucleotide is 38 nucleosides in length. In some embodiments, an oligonucleotide is 39 nucleosides in length. In some embodiments, an oligonucleotide is 40 nucleosides in length.
  • each nucleoside counted in an oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in an oligonucleotide length independently comprises A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G or U. [0048] Optionally Substituted: As described herein, compounds, e.g., oligonucleotides, of the disclosure may contain optionally substituted and/or substituted moieties.
  • substituted means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • an optionally substituted group is unsubstituted. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below.
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 ) 0-2 R • , -(haloR • ), -(CH 2 ) 0 - 2 OH, -(CH 2 ) 0-2 OR • , -(CH 2 ) 0-2 CH(OR • ) 2 ; -O(haloR • ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R • , -(CH 2 ) 0 _ 2 C(O)OH, -(CH 2 ) 0-2 C(O)OR • , -(CH 2 ) 0-2 SR • , -(CH 2 ) 0-2 SH, -(CH 2 ) 0-2 NH 2 , -(CH 2 ) 0-2 NHR • , -(CH 2 )
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: - O(CR* 2 ) 2-3 O-, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, and aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Suitable substituents on the aliphatic group of R* are independently halogen, -R • , -(haloR • ), - OH, -OR • , -O(halo R • ), -CN, -C(O)OH, -C(O)OR • , -NH 2 , -NHR • , -NR • 2 , or -NO 2 , wherein each R • is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • suitable substituents on a substitutable nitrogen are independently -R ⁇ -NR ⁇ 2 -C(O)R ⁇ , -C(O)OR ⁇ , -C(O)C(O)R ⁇ , -C(O)CH 2 C(O)R ⁇ , -S(O) 2 R ⁇ , -S(O) 2 NR ⁇ 2 , -C(S)NR ⁇ 2 , - C(NH)NR ⁇ 2 .
  • each R ⁇ is independently hydrogen, C 1-6 aliphatic which may be substituted as defined below, unsubstituted OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R ⁇ taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • Suitable substituents on the aliphatic group of R are independently halogen, -R • , -(haloR • ), - OH, OR • , -O(haloR • ), CN, C(O)OH, C(O)OR • , -NH 2 , -NHR • , NR • 2 , or -NO 2 , wherein each R • is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently
  • Partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
  • compositions or vehicles which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydrox
  • compositions that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
  • pharmaceutically acceptable salt include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palm
  • a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R) 3 , wherein each R is independently defined and described in the present disclosure) salt.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • a pharmaceutically acceptable salt is a sodium salt.
  • a pharmaceutically acceptable salt is a potassium salt.
  • a pharmaceutically acceptable salt is a calcium salt.
  • pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
  • a provided compound comprises more than one acid groups, for example, an oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and/or modified internucleotidic linkages).
  • a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different.
  • all ionizable hydrogen e.g., in an aqueous solution with a pKa no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3 in the acidic groups are replaced with cations.
  • each phosphorothioate and phosphate group independently exists in its salt form (e.g., if sodium salt, ⁇ O ⁇ P(O)(SNa) ⁇ O ⁇ and ⁇ O ⁇ P(O)(ONa) ⁇ O ⁇ , respectively).
  • each phosphorothioate and phosphate internucleotidic linkage independently exists in its salt form (e.g., if sodium salt, ⁇ O ⁇ P(O)(SNa) ⁇ O ⁇ and ⁇ O ⁇ P(O)(ONa) ⁇ O ⁇ , respectively).
  • a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide.
  • a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, exists as a salt form (all sodium salt).
  • Protecting group The term “protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
  • Suitable amino–protecting groups include methyl carbamate, ethyl carbamante, 9–fluorenylmethyl carbamate (Fmoc), 9–(2–sulfo)fluorenylmethyl carbamate, 9–(2,7– dibromo)fluoroenylmethyl carbamate, 2,7–di–t–butyl–[9–(10,10–dioxo–10,10,10,10– tetrahydrothioxanthyl)]methyl carbamate (DBD–Tmoc), 4–methoxyphenacyl carbamate (Phenoc), 2,2,2– trichloroethyl carbamate (Troc), 2–trimethylsilylethyl carbamate (Teoc), 2–phenylethyl carbamate (hZ), 1–(1– adamantyl)–1–methylethyl carbamate (Adpoc), 1,1–dimethyl–2–haloe
  • Suitably protected carboxylic acids further include, but are not limited to, silyl–, alkyl–, alkenyl– , aryl–, and arylalkyl–protected carboxylic acids.
  • suitable silyl groups include trimethylsilyl, triethylsilyl, t–butyldimethylsilyl, t–butyldiphenylsilyl, triisopropylsilyl, and the like.
  • suitable alkyl groups include methyl, benzyl, p–methoxybenzyl, 3,4–dimethoxybenzyl, trityl, t–butyl, tetrahydropyran– 2–yl.
  • suitable alkenyl groups include allyl.
  • suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl.
  • suitable arylalkyl groups include optionally substituted benzyl (e.g., p–methoxybenzyl (MPM), 3,4–dimethoxybenzyl, O–nitrobenzyl, p–nitrobenzyl, p–halobenzyl, 2,6–dichlorobenzyl, p–cyanobenzyl), and 2– and 4–picolyl.
  • Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t–butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p– methoxybenzyloxymethyl (PMBM), (4–methoxyphenoxy)methyl (p–AOM), guaiacolmethyl (GUM), t– butoxymethyl, 4–pentenyloxymethyl (POM), siloxymethyl, 2–methoxyethoxymethyl (MEM), 2,2,2– trichloroethoxymethyl, bis(2–chloroethoxy)methyl, 2–(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3–bromotetrahydropyranyl, tetrahydrothiopyranyl, 1–methoxycyclohexyl, 4– methoxytetrahydropyr
  • the protecting groups include methylene acetal, ethylidene acetal, 1–t– butylethylidene ketal, 1–phenylethylidene ketal, (4–methoxyphenyl)ethylidene acetal, 2,2,2– trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p–methoxybenzylidene acetal, 2,4–dimethoxybenzylidene ketal, 3,4– dimethoxybenzylidene acetal, 2–nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1–methoxyethy
  • a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6- dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trich
  • each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t- butyldimethylsilyl, t- butyldiphenylsilyl and 4,4'-dimethoxytrityl.
  • the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group.
  • a phosphorous linkage protecting group is a group attached to the phosphorous linkage (e.g., an internucleotidic linkage) throughout oligonucleotide synthesis.
  • a protecting group is attached to a sulfur atom of an phosphorothioate group. In some embodiments, a protecting group is attached to an oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, a protecting group is attached to an oxygen atom of the internucleotide phosphate linkage.
  • a protecting group is 2- cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2- (p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4- methylthio-l-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl- N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2- trifluoroacetyl)amino]butyl.
  • Subject refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and/or susceptible to a disease, disorder and/or condition.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.
  • a subject is a human.
  • a subject may be suffering from and/or susceptible to a disease, disorder and/or condition.
  • sugar refers to a monosaccharide or polysaccharide in closed and/or open form.
  • sugars are monosaccharides.
  • sugars are polysaccharides.
  • Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties.
  • the term “sugar” also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol nucleic acid (“GNA”), etc.
  • a sugar also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars.
  • a sugar is a RNA or DNA sugar (ribose or deoxyribose).
  • a sugar is a modified ribose or deoxyribose sugar, e.g., 2’-modified, 5’-modified, etc.
  • modified sugars when used in oligonucleotides and/or nucleic acids, modified sugars may provide one or more desired properties, activities, etc.
  • a sugar is optionally substituted ribose or deoxyribose.
  • a “sugar” refers to a sugar unit in an oligonucleotide or a nucleic acid.
  • an individual who is susceptible to a disease, disorder and/or condition may exhibit symptoms of the disease, disorder and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not exhibit symptoms of the disease, disorder and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • therapeutic agent in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject.
  • a desired effect e.g., a desired biological, clinical, or pharmacological effect
  • an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
  • an appropriate population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition.
  • an appropriate population is a population of model organisms.
  • an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy.
  • a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a disease, disorder, and/or condition in a subject when administered to the subject in an effective amount.
  • a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans.
  • a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans.
  • a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.
  • therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • Wild-type As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles). [0071] As those skilled in the art will appreciate, methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds.
  • provided compounds e.g., oligonucleotides
  • “one or more” is 1-200, 1-150, 1-100, 1- 90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
  • “one or more” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three.
  • “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six.
  • oligonucleotides of the present disclosure target calpain-2 and can hybridize with a calpain-2 transcript, e.g., a calpain-2 mRNA.
  • provided technologies reduce levels of calpain-2 transcripts and/or products thereof.
  • Use of naturally occurring nucleic acids is limited, for example, by their susceptibility to endo- and exo- nucleases.
  • various synthetic counterparts have been developed to circumvent these shortcomings and/or to further improve various properties and activities.
  • provided oligonucleotides comprise various chemical modifications, e.g., nucleobase modifications, sugar modifications, internucleotidic linkage modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties and/or activities.
  • an oligonucleotide comprises one or more features described herein, e.g., base sequence, length, wing, core, activity, etc. In some embodiments, an oligonucleotide has a base sequence described herein, and/or a wing-core-wing structure as described herein.
  • Base Sequences [0075] Base sequences of various oligonucleotides are of sufficient lengths so that they can form duplexes with complementary sequences in target nucleic acids for one or more biological functions. In some embodiments, oligonucleotides specifically target their target nucleic acids.
  • the base sequence of a provided oligonucleotide is or comprises a sequence that is complementary to a portion in a target nucleic acid (a “target portion”), e.g., a calpain-2 gene or a transcript thereof.
  • a target portion e.g., a calpain-2 gene or a transcript thereof.
  • a sequence complementary to a target portion is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleobases in length.
  • a target portion is or comprises a characteristic portion of a nucleic acid sequence (e.g., of a calpain-2 gene or a transcript thereof) which characteristic portion defines the nucleic acid sequence over others in a relevant organism; for example, the characteristic portion is not in other genomic nucleic acid sequences (e.g., genes) or transcripts thereof in a relevant organism (e.g., for human calpain-2, its characteristic portion is not in other human nucleic acid sequences or transcripts thereof).
  • a characteristic portion of a nucleic acid sequence e.g., of a calpain-2 gene or a transcript thereof
  • a characteristic portion of a transcript defines that transcript over other transcripts in a relevant organism; for example, in some embodiments, the characteristic portion is not in transcripts that are transcribed from a different nucleic acid sequence (e.g., a different gene).
  • transcript variants from a nucleic acid sequence e.g., mRNA variants of a gene
  • a characteristic portion in a transcript defines the transcript from other transcript(s) of the same nucleic acid sequence (e.g., a gene) and/or other alleles of the nucleic acid sequence.
  • a characteristic portion defines a particular allele (and/or transcripts thereof) over other allele(s) (and/or transcripts thereof).
  • a characteristic portion comprises sequences that are separated in a nucleic acid.
  • a characteristic portion is a contiguous stretch of nucleobases in a nucleic acid (a “characteristic sequence”).
  • a characteristic portion or sequence may have various numbers of nucleobases.
  • an oligonucleotide comprises a sequence that is identical or complementary to a characteristic portion of a nucleic acid. In some embodiments, an oligonucleotide comprises a sequence that is identical or complementary to a characteristic portion of a calpain-2 transcript. In some embodiments, an oligonucleotide comprises a sequence that is complementary to a characteristic portion of a calpain-2 transcript. In some embodiments, the base sequence of an oligonucleotide is identical or complementary to a characteristic portion of a nucleic acid. In some embodiments, the base sequence of an oligonucleotide is identical or complementary to a characteristic portion of a calpain-2 transcript.
  • the base sequence of an oligonucleotide is complementary to a characteristic portion of a calpain-2 transcript.
  • a characteristic portion is a characteristic sequence.
  • a characteristic sequence of a calpain-2 transcript is or comprises a complementary sequence of the sequence of an oligonucleotide in Table 1.
  • a characteristic sequence is or comprises GGAAGCCUACAGAAACUGAU, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises AUCAACUGAACCAGUAUGCC, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises AACCACUGCCUGACCUGAGC, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises AGCUCUGCAAAAAGGCUCUC, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises ACUAGAGGCACAGAGCUGGA, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises ACAACUGCCCAAGCUGGAAC, wherein each U may be independently replaced with T.
  • a characteristic sequence is or comprises CAGCCAAGGACUAAGCUUCC, wherein each U may be independently replaced with T.
  • an oligonucleotide can hybridize to a region of a nucleic acid.
  • oligonucleotides that can specifically hybridize (e.g., through sequence complementarity) to certain region(s) of a nucleic acid can more effectively reduce levels of the nucleic acid than oligonucleotides that specifically hybridize (e.g., through sequence complementarity) to one or more reference regions of the nucleic acid.
  • a region has a length of about 20-200 (e.g., about 20-150, 20-100, 30- 200, 30-150, 40-200, 40-150, 50-100, or about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200) nucleobases.
  • a region has a length of about 30 nucleobases.
  • a region has a length of about 40 nucleobases.
  • a region has a length of about 50 nucleobases.
  • a region has a length of about 60 nucleobases.
  • a region has a length of about 70 nucleobases. In some embodiments, a region has a length of about 80 nucleobases. In some embodiments, a region has a length of about 90 nucleobases. In some embodiments, a region has a length of about 100 nucleobases. In some embodiments, a region has a length of about 120 nucleobases. In some embodiments, a region has a length of about 150 nucleobases. In some embodiments, a region has a length of about 200 nucleobases. In some embodiments, a region comprises a base sequence of an oligonucleotide in Table 1, which in some embodiments, is in the middle of a region.
  • a region is or comprises GGAAGCCUACAGAAACUGAU.
  • a region is or comprises AUCAACUGAACCAGUAUGCC.
  • a region is or comprises AACCACUGCCUGACCUGAGC.
  • a region is or comprises AGCUCUGCAAAAAGGCUCUC.
  • a region is or comprises ACUAGAGGCACAGAGCUGGA.
  • a region is or comprises ACAACUGCCCAAGCUGGAAC.
  • a region is or comprises CAGCCAAGGACUAAGCUUCC.
  • a reference region is or comprises UCCCUCACCUUGAAUGAAGA.
  • base sequences of oligonucleotides comprise or consist of about 10-50 (e.g., about 15-50, 16-50, 17-50, 18-50, 19-50, 20-50, 15-30, 20-30, 15-25 or 20-25, or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45; in some embodiments, at least about 15; in some embodiments, at least about 16; in some embodiments, at least about 17; in some embodiments, at least about 18; in some embodiments, at least about 19; in some embodiments, at least about 20; in some embodiments, at least about 21; in some embodiments, at least about 22; in some embodiments, at least about 23; in some embodiments, at least about 24; in some embodiments, at least about 25) contiguous bases that are identical to or complementary to a base sequence of equal length in a calpain-2 gene or a transcript (e.g., mRNA) thereof.
  • a transcript e.g., mRNA
  • a base sequence of an oligonucleotide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to a target sequence in a calpain-2 transcript. In some embodiments, a base sequence of an oligonucleotide is fully complementary to a target sequence in a calpain-2 transcript.
  • the base sequence of an oligonucleotide has about 80% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently replaced with U and vice versa. In some embodiments, the base sequence of an oligonucleotide has about 85% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently replaced with U and vice versa. In some embodiments, the base sequence of an oligonucleotide has about 90% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide has about 95% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises a continuous span of about 15 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises a continuous span of about 16 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises a continuous span of about 17 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa. In some embodiments, the base sequence of an oligonucleotide comprises a continuous span of about 18 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa. In some embodiments, the base sequence of an oligonucleotide comprises a continuous span of about 19 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises a continuous span of about 20 or more bases of an oligonucleotide disclosed in a Table 1, wherein each T can be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises the base sequence of an oligonucleotide in Table 1, wherein each T may be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide is the base sequence of an oligonucleotide in Table 1, wherein each T may be independently replaced with U and vice versa.
  • the base sequence of an oligonucleotide comprises ATCAGTTTCTGTAGGCTTCC, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises GGCATACTGGTTCAGTTGAT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises GCTCAGGTCAGGCAGTGGTT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises GAGAGCCTTTTTGCAGAGCT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises TCCAGCTCTGTGCCTCTAGT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises GTTCCAGCTTGGGCAGTTGT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide comprises GGAAGCTTAGTCCTTGGCTG, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is ATCAGTTTCTGTAGGCTTCC, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is GGCATACTGGTTCAGTTGAT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is GCTCAGGTCAGGCAGTGGTT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is GAGAGCCTTTTTGCAGAGCT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is TCCAGCTCTGTGCCTCTAGT, wherein each T may be independently replaced with U.
  • the base sequence of an oligonucleotide is GTTCCAGCTTGGGCAGTTGT, wherein each T may be independently replaced with U. In some embodiments, the base sequence of an oligonucleotide is GGAAGCTTAGTCCTTGGCTG, wherein each T may be independently replaced with U. [0084] In some embodiments, the base sequence of an oligonucleotide comprises ATCAGTTTCTGTAGGCTTCC. In some embodiments, the base sequence of an oligonucleotide comprises GGCATACTGGTTCAGTTGAT. In some embodiments, the base sequence of an oligonucleotide comprises GCTCAGGTCAGGCAGTGGTT.
  • the base sequence of an oligonucleotide comprises GAGAGCCTTTTTGCAGAGCT. In some embodiments, the base sequence of an oligonucleotide comprises TCCAGCTCTGTGCCTCTAGT. In some embodiments, the base sequence of an oligonucleotide comprises GTTCCAGCTTGGGCAGTTGT. In some embodiments, the base sequence of an oligonucleotide comprises GGAAGCTTAGTCCTTGGCTG. In some embodiments, the base sequence of an oligonucleotide is ATCAGTTTCTGTAGGCTTCC. In some embodiments, the base sequence of an oligonucleotide is GGCATACTGGTTCAGTTGAT.
  • the base sequence of an oligonucleotide is GCTCAGGTCAGGCAGTGGTT. In some embodiments, the base sequence of an oligonucleotide is GAGAGCCTTTTTGCAGAGCT. In some embodiments, the base sequence of an oligonucleotide is TCCAGCTCTGTGCCTCTAGT. In some embodiments, the base sequence of an oligonucleotide is GTTCCAGCTTGGGCAGTTGT. In some embodiments, the base sequence of an oligonucleotide is GGAAGCTTAGTCCTTGGCTG.
  • oligonucleotides can be of various lengths to provide desired properties and/or activities for various uses. Many technologies for assessing, selecting and/or optimizing oligonucleotide length are available in the art and can be utilized in accordance with the present disclosure. As demonstrated herein, in many embodiments, provided oligonucleotides are of suitable lengths to hybridize with their targets and reduce levels of their targets and/or a product thereof. In some embodiments, an oligonucleotide is long enough to recognize a target nucleic acid (e.g., a calpain-2 mRNA).
  • a target nucleic acid e.g., a calpain-2 mRNA
  • an oligonucleotide is sufficiently long to distinguish between a target nucleic acid and other nucleic acids (e.g., a nucleic acid having a base sequence which is not a calpain-2 sequence) to reduce off- target effects.
  • an oligonucleotide is sufficiently short to reduce complexity of manufacture or production and to reduce cost of products.
  • the base sequence of an oligonucleotide is about 10-100 nucleobases in length. In some embodiments, a base sequence is about 10-50 nucleobases in length. In some embodiments, a base sequence is about 15-50 nucleobases in length.
  • a base sequence is about 15-30 nucleobases in length. In some embodiments, a base sequence is about 15-25 nucleobases in length. In some embodiments, a base sequence is about 15-22 nucleobases in length. In some embodiments, a base sequence is about 18-22 nucleobases in length. In some embodiments, a base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases in length. In some embodiments, a base sequence is at least about 12 nucleobases in length. In some embodiments, a base sequence is at least about 13 nucleobases in length. In some embodiments, a base sequence is at least about 14 nucleobases in length.
  • a base sequence is at least about 15 nucleobases in length. In some embodiments, a base sequence is at least about 16 nucleobases in length. In some embodiments, a base sequence is at least about 17 nucleobases in length. In some embodiments, a base sequence is at least about 18 nucleobases in length. In some embodiments, a base sequence is at least about 19 nucleobases in length. In some embodiments, a base sequence is at least about 20 nucleobases in length. In some embodiments, a base sequence is at least about 21 nucleobases in length. In some embodiments, a base sequence is at least about 22 nucleobases in length.
  • a base sequence is at least about 23 nucleobases in length. In some embodiments, a base sequence is at least about 24 nucleobases in length. In some embodiments, a base sequence is at least about 25 nucleobases in length. In some embodiments, a base sequence is about 15 nucleobases in length. In some embodiments, a base sequence is about 16 nucleobases in length. In some embodiments, a base sequence is about 17 nucleobases in length. In some embodiments, a base sequence is about 18 nucleobases in length. In some embodiments, a base sequence is about 19 nucleobases in length. In some embodiments, a base sequence is about 20 nucleobases in length.
  • a base sequence is about 21 nucleobases in length. In some embodiments, a base sequence is about 22 nucleobases in length. In some embodiments, a base sequence is about 23 nucleobases in length. In some embodiments, a base sequence is about 24 nucleobases in length. In some embodiments, a base sequence is about 25 nucleobases in length. In some other embodiments, a base sequence is about at least about 30 nucleobases in length. In some embodiments, each nucleobase independently comprises an optionally substituted monocyclic, bicyclic or polycyclic ring wherein at least one ring atom is nitrogen.
  • each nucleobase is independently optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or an optionally substituted tautomer of adenine, cytosine, guanosine, thymine, or uracil.
  • Nucleobases may be utilized in provided oligonucleotides in accordance with the present disclosure.
  • a nucleobase is a natural nucleobase, the most commonly occurring ones being A, T, C, G and U.
  • a nucleobase is a modified nucleobase in that it is not A, T, C, G or U.
  • a nucleobase is optionally substituted A, T, C, G or U, or a substituted tautomer of A T, C, G or U.
  • a nucleobase is optionally substituted A, T, C, G or U, e.g., 5mC, 5-hydroxymethyl C, etc.
  • a nucleobase is alkyl-substituted A, T, C, G or U.
  • a nucleobase is A.
  • a nucleobase is T.
  • a nucleobase is C.
  • a nucleobase is G.
  • a nucleobase is U.
  • a nucleobase is 5mC. In some embodiments, a nucleobase is substituted A, T, C, G or U. In some embodiments, a nucleobase is a substituted tautomer of A, T, C, G or U. In some embodiments, substitution protects certain functional groups in nucleobases to minimize undesired reactions during oligonucleotide synthesis. Suitable technologies for nucleobase protection in oligonucleotide synthesis are widely known in the art and may be utilized in accordance with the present disclosure. In some embodiments, modified nucleobases improves properties and/or activities of oligonucleotides.
  • 5mC may be utilized in place of C to modulate certain undesired biological effects, e.g., immune responses.
  • a substituted nucleobase having the same hydrogen-bonding pattern is treated as the same as the unsubstituted nucleobase, e.g., 5mC may be treated the same as C [e.g., an oligonucleotide having 5mC in place of C (e.g., AT5mCG) is considered to have the same base sequence as an oligonucleotide having C at the corresponding location(s) (e.g., ATCG)].
  • an oligonucleotide comprises one or more A, T, C, G or U. In some embodiments, an oligonucleotide comprises one or more optionally substituted A, T, C, G or U. In some embodiments, an oligonucleotide comprises one or more 5-methylcytosine (5mC), 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine. In some embodiments, an oligonucleotide comprises one or more 5mC.
  • each nucleobase in an oligonucleotide is independently selected from optionally substituted A, T, C, G and U, and optionally substituted tautomers of A, T, C, G and U.
  • each nucleobase in an oligonucleotide is independently optionally protected A, T, C, 5mC, G and U.
  • each nucleobase in an oligonucleotide is optionally substituted A, T, C, G or U.
  • each nucleobase in an oligonucleotide is selected from the group consisting of A, T, C, G, U, and 5mC.
  • a nucleobase is optionally substituted 2AP or DAP. In some embodiments, a nucleobase is optionally substituted 2AP. In some embodiments, a nucleobase is optionally substituted DAP. In some embodiments, a nucleobase is 2AP. In some embodiments, a nucleobase is DAP. [0090] In some embodiments, a nucleobase is a natural nucleobase or a modified nucleobase derived from a natural nucleobase.
  • Examples include uracil, thymine, adenine, cytosine, and guanine optionally having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products).
  • modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al.
  • a provided oligonucleotide comprises one or more 5-methylcytosine.
  • the present disclosure provides an oligonucleotide whose base sequence is disclosed herein, e.g., in Table 1, wherein each T may be independently replaced with U and vice versa, and each cytosine is optionally and independently replaced with 5-methylcytosine or vice versa.
  • 5mC may be treated as C with respect to base sequence of an oligonucleotide - such oligonucleotide comprises a nucleobase modification at the C position (e.g., see various oligonucleotides in Table 1 or A2).
  • nucleobases, sugars and internucleotidic linkages are non-modified.
  • a modified nucleobase is a modified nucleobase known in the art, e.g., WO2017/210647.
  • modified nucleobases are expanded-size nucleobases in which one or more aryl and/or heteroaryl rings, such as phenyl rings, have been added.
  • Nucleobases may be protected during oligonucleotide synthesis. Various protection technologies are available and can be utilized in accordance with the present disclosure.
  • a modified nucleobase is 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, or N-2, N-6 and O-6 substituted purines.
  • a modified nucleobase is selected form 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2- propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( ⁇ C ⁇ C-CH 3 ) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4- thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5- bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7
  • a modified nucleobases is a tricyclic pyrimidine, such as l,3-diazaphenoxazine- 2-one, l,3-diazaphenothiazine-2-one or 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2- one (G-clamp).
  • a modified nucleobases is one in which a purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine or 2- pyridone. [0095] In some embodiments, a modified nucleobase is substituted.
  • a modified nucleobase is substituted such that it contains, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other protein or peptides.
  • a modified nucleobase is a “universal base” that is not a nucleobase in the most classical sense, but that functions similarly to a nucleobase.
  • a universal base is 3-nitropyrrole.
  • nucleosides that can be utilized in provided technologies comprise modified nucleobases and/or modified sugars, e.g., 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2’- O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2’-O-methylpseudouridine; beta,D-galactosylqueosine; 2’-O-methylguanosine; N 6 - isopentenyladenosine; 1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine; l-methylinosine; 2,2- dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N 7 -methylguanosine; 3-methyl-cytidine; 5- methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosethyl, 5-form
  • a nucleobase e.g., a modified nucleobase comprises one or more biomolecule binding moieties such as e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties.
  • a nucleobase is 5-bromouracil, 5-iodouracil, or 2,6- diaminopurine.
  • a nucleobase comprises substitution with a fluorescent or biomolecule binding moiety.
  • a substituent is a fluorescent moiety.
  • a substituent is biotin or avidin.
  • a nucleobase is one described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/0127733, US 10450568, US 2019/0077817, US 2019/0249173, US 2019/0375774, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/032612, the nucleobase of each of which is incorporated herein by reference.
  • sugars including modified sugars, can be utilized in accordance with the present disclosure.
  • the present disclosure provides sugar modifications and patterns thereof optionally in combination with other structural elements (e.g., nucleobase modifications and patterns thereof, internucleotidic linkage modifications and patterns thereof, etc.) that when incorporated into oligonucleotides can provide improved properties and/or activities.
  • nucleosides comprise ribose sugars (e.g., in RNA) or deoxyribose sugars (e.g., in DNA) linked to the nucleobases adenosine (A), cytosine (C), guanine (G), thymine (T) or uracil (U).
  • ribose sugars e.g., in RNA
  • deoxyribose sugars e.g., in DNA linked to the nucleobases adenosine (A), cytosine (C), guanine (G), thymine (T) or uracil (U).
  • a sugar e.g., various sugars in many oligonucleotides in Table 1 (unless otherwise notes), is a natural DNA sugar (in DNA nucleic acids or oligonucleotides, having the structure of , wherein a nucleobase is attached to the 1’ position, and the 3’ and 5’ positions are connected to internucleotidic linkages (as appreciated by those skilled in the art, if at the 5’-end of an oligonucleotide, the 5’ position may be connected to a 5’-end group (e.g., ⁇ OH), and if at the 3’-end of an oligonucleotide, the 3’ position may be connected to a 3’-end group (e.g., ⁇ OH).
  • a 5’-end group e.g., ⁇ OH
  • 3’-end group e.g., ⁇ OH
  • a sugar is a natural RNA sugar (in RNA nucleic acids or oligonucleotides, having the structure of , wherein a nucleobase is attached to the 1’ position, and the 3’ and 5’ positions are connected to internucleotidic linkages (as appreciated by those skilled in the art, if at the 5’-end of an oligonucleotide, the 5’ position may be connected to a 5’-end group (e.g., ⁇ OH), and if at the 3’-end of an oligonucleotide, the 3’ position may be connected to a 3’-end group (e.g., ⁇ OH).
  • a sugar is a modified sugar in that it is not a natural DNA sugar or a natural RNA sugar.
  • modified sugars may provide improved stability and/or affinity.
  • modified sugars can be utilized to alter and/or optimize one or more hybridization characteristics.
  • modified sugars can be utilized to alter and/or optimize target recognition.
  • modified sugars can be utilized to optimize Tm.
  • modified sugars can be utilized to improve oligonucleotide activities.
  • Sugars can be bonded to internucleotidic linkages at various positions. As non-limiting examples, internucleotidic linkages can be bonded to the 2’, 3’, 4’ or 5’ positions of sugars.
  • a sugar is an optionally substituted natural DNA or RNA sugar. In some embodiments, a sugar is optionally substituted . In some embodiments, the 2’ position is optionally substituted. In some embodiments, a sugar is .
  • a sugar has the structure of or , wherein each of R 1s , R 2s , R 3s , R 4s , and R 5s is independently ⁇ H, a suitable substituent or suitable sugar modification (e.g., those described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017/062862, WO 2018/067973, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/022473, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO2019/032612, WO 2019/055951, and/or WO 2019/075357, the substituents, sugar modifications, descriptions of R 1s , R 2s , R 3s
  • each of R 1s , R 2s , R 3s , R 4s , and R 5s is independently ⁇ F, ⁇ Cl, ⁇ Br, ⁇ I, ⁇ CN, ⁇ N 3 , ⁇ NO, ⁇ NO 2 , ⁇ L s ⁇ R’, ⁇ L s ⁇ OR’, ⁇ L s ⁇ SR’, ⁇ L s ⁇ N(R’) 2 , ⁇ O ⁇ L s ⁇ OR’, ⁇ O ⁇ L s ⁇ SR’, or ⁇ O ⁇ L s ⁇ N(R’) 2 , wherein each R’ is independently ⁇ H or an optionally substituted group selected from C 1-10 aliphatic, C 6-14 aryl, C 1-10 heteroaliphatic having 1-5 heteroatoms, 5-10membered heteroaryl having 1-5 heteroatoms and 3-10 membered heterocyclyl having 1-4 heteroatoms, or two or more R’ groups are taken together with their intervening atoms to
  • L s is a covalent bond or optionally substituted bivalent C 1-6 aliphatic or heteroaliphatic having 1-4 heteroatoms.
  • a sugar has the structure of 4s .
  • R is ⁇ H.
  • a sugar has the structure of 2s , wherein R is ⁇ H, halogen, or ⁇ OR, wherein R is optionally substituted C 1-6 aliphatic.
  • R 2s is ⁇ H.
  • R 2s is ⁇ F.
  • R 2s is ⁇ OMe.
  • R 2s is ⁇ OCH 2 CH 2 OMe.
  • a sugar has the structure of 2s 4s , wherein R and R are taken together to form ⁇ L s ⁇ , wherein L s is a covalent bond or optionally substituted bivalent C 1-6 aliphatic or heteroaliphatic having 1-4 heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen or sulfur). In some embodiments, L s is optionally substituted C2 ⁇ O ⁇ CH 2 ⁇ C4. In some embodiments, L s is C2 ⁇ O ⁇ CH 2 ⁇ C4. In some embodiments, L s is C2 ⁇ O ⁇ (R)-CH(CH 2 CH 3 ) ⁇ C4.
  • L s is C2 ⁇ O ⁇ (S)-CH(CH 2 CH 3 ) ⁇ C4.
  • a modified sugar contains one or more substituents at the 2’ position ( (typically one substituent, and often at the axial position or R 2s ) independently selected from –F; –CF 3 , –CN, –N 3 , –NO, –NO 2 , –OR’, –SR’, or –N(R’) 2 , wherein each R’ is independently described in the present disclosure, and in some embodiments, optionally substituted C 1-10 aliphatic; –O–(C 1 –C 10 alkyl), –S–(C 1 –C 10 alkyl), –NH–(C 1 –C 10 alkyl), or –N(C 1 –C 10 alkyl) 2 ; –O–(C 2 –C 10 alkenyl), –S–(C 2 –C 10 alkenyl),
  • a substituent is –O(CH 2 ) n OCH 3 , –O(CH 2 ) n NH 2 , MOE, DMAOE, or DMAEOE, wherein n is from 1 to about 10.
  • a modified sugar is a natural RNA sugar whose 2’-OH is replaced with a group selected from–F, –CF 3 , –CN, –N 3 , –NO, –NO 2 , –OR’, –SR’, or –N(R’) 2 , wherein each R’ is independently described in the present disclosure; –O–(C 1 –C 10 alkyl), –S–(C 1 –C 10 alkyl), –NH–(C 1 –C 10 alkyl), or –N(C 1 –C 10 alkyl) 2 ; –O–(C 2 –C 10 alkenyl), –S–(C 2 –C 10 alkenyl),
  • the 2’–OH is replaced with –H (deoxyribose). In some embodiments, the 2’–OH is replaced with –F. In some embodiments, the 2’–OH is replaced with –OR’. In some embodiments, the 2’–OH is replaced with –OMe. In some embodiments, the 2’–OH is replaced with –OCH 2 CH 2 OMe.
  • a sugar modification is a 2’-modification. In some embodiments, a 2’- modification is a 2’-OR s modification. In some embodiments, R s is optionally substituted C 1-4 aliphatic. In some embodiments, R s is optionally substituted C 1-6 alkyl.
  • a modification is 2’ ⁇ OMe. In some embodiments, a modification is 2’-MOE. In some embodiments, a 2’-modification is S-cEt. In some embodiments, a modified sugar is an LNA sugar. In some embodiments, a 2’-modification is ⁇ F. [00107] In some embodiments, a sugar modification replaces a sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, e.g., those in morpholino, glycol nucleic acids, PNA, etc., and may be utilized in accordance with the present disclosure.
  • one or more sugars of an oligonucleotide are independently modified.
  • each sugar of an oligonucleotide or a portion thereof is independently modified.
  • a modified sugar comprises a 2’-modification.
  • each modified sugar independently comprises a 2’-modification.
  • a 2’-modification is 2’- OR s , wherein R s is optionally substituted C 1-6 aliphatic.
  • a 2’-modification is a 2’-OMe modification.
  • a 2’-modification is a 2’-MOE modification.
  • a 2’-modification is an LNA sugar modification. In some embodiments, a 2’-modification is 2’-F. In some embodiments, each sugar modification is independently a 2’-modification. In some embodiments, each sugar modification is independently a 2’-OR s modification. In some embodiments, each sugar modification is independently 2’-OR s , wherein R s is optionally substituted C 1-6 alkyl. In some embodiments, each sugar modification is 2’-OMe. In some embodiments, each sugar modification is 2’-MOE. In some embodiments, each sugar modification is independently 2’-OMe or 2’-MOE. In some embodiments, each sugar modification is independently 2’-OMe, 2’-MOE, or a LNA sugar.
  • a sugar is one described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/0127733, US 10450568, US 2019/0077817, US 2019/0249173, US 2019/0375774, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/032612, the sugars of each of which is incorporated herein by reference.
  • oligonucleotides comprise base modifications, sugar modifications, and/or internucleotidic linkage modifications.
  • Various internucleotidic linkages can be utilized in accordance with the present disclosure to link units comprising nucleobases, e.g., nucleosides.
  • oligonucleotides comprise both one or more modified internucleotidic linkages and one or more natural phosphate linkages.
  • natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure of ⁇ OP(O)(OH)O ⁇ , connect sugars in the nucleosides in DNA and RNA, and may be in various salt forms, for example, at physiological pH (about 7.4), natural phosphate linkages are predominantly exist in salt forms with the anion being ⁇ OP(O)(O ⁇ )O ⁇ .
  • a modified internucleotidic linkage, or a non-natural phosphate linkage is an internucleotidic linkage that is not natural phosphate linkage or a salt form thereof. Modified internucleotidic linkages, depending on their structures, may also be in their salt forms.
  • an oligonucleotide comprises an internucleotidic linkage which is a modified internucleotidic linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroamidate, thiophosphate, 3’-thiophosphate, or 5’-thiophosphate.
  • an internucleotidic linkage is described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020/0056173, US 2018/0216107, US 2019/0127733, US 10450568, US 2019/0077817, US 2019/0249173, US 2019/0375774, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/032612, the internucleotidic linkages of each of which is incorporated herein by reference.
  • an internucleotidic linkage is described in U.S. Pat. Nos. 3687808, 4469863, 4476301, 5177195, 5023243, 5034506, 5166315, 5185444, 5188897, 5214134, 5216141, 5235033, 5264423, 5264564, 5276019, 5278302, 5286717, 5321131, 5399676, 5405938, 5405939, 5434257, 5453496, 5455233, 5466677, 5466677, 5470967, 5476925, 5489677, 5519126, 5536821, 5541307, 5541316, 5550111, 5561225, 5563253, 5571799, 5587361, 5596086, 5602240, 5608046, 5610289, 5618704, 5623070, 5625050, 5633360, 564562, 5663312, 5677437, 5677439, 6160109, 6239265, 60
  • an oligonucleotide comprises one or more modified internucleotidic linkages.
  • each modified internucleotidic linkage is independently a phosphorothioate internucleotidic linkage.
  • about 25% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 50% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 60% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 70% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages.
  • about 75% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 80% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 85% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, about 90% or more of all internucleotidic linkages are independently phosphorothioate internucleotidic linkages.
  • each internucleotidic linkage bonded to a natural DNA sugar is independently a phosphorothioate internucleotidic linkage.
  • each internucleotidic linkage in an oligonucleotide is independently a phosphorothioate internucleotidic linkage.
  • an oligonucleotide comprises one or more natural phosphate linkages.
  • each natural phosphate linkage independently bonds to at least one modified sugar.
  • each sugar bonded to a natural phosphate linkage is independently a modified sugar. In some embodiments, each sugar bonded to a natural phosphate linkage is independently a 2’-OR s modified sugar or a bicyclic sugar (e.g., a LNA sugar). In some embodiments, each sugar bonded to a natural phosphate linkage is independently a 2’-OR s modified sugar. In some embodiments, each sugar bonded to a natural phosphate linkage is independently a 2’-MOE modified sugar. Wings and Cores [00117] In some embodiments, an oligonucleotide comprises or consists of a 5’-wing-core-wing-3’ structure.
  • Wings and cores can independently be of various suitable lengths. In some embodiments, there are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleobases independently in a wing or core.
  • each nucleobase independently comprises an optionally substituted monocyclic, bicyclic or polycyclic ring, which ring has at least one nitrogen ring atom; in some embodiments, each nucleobase is independently optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U. In some embodiments, the number of nucleobases in a wing is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the number is 1 for a wing. In some embodiments, the number is 2 for a wing. In some embodiments, the number is 3 for a wing. In some embodiments, the number is 4 for a wing. In some embodiments, the number is 5 for a wing. In some embodiments, the number is 6 for a wing. In some embodiments, the number is 7 for a wing. In some embodiments, the number is 8 for a wing. In some embodiments, the number is 9 for a wing. In some embodiments, the number is 10 for a wing. In some embodiments, in a wing of a wing-core-wing structure, the two wings are of the same length.
  • the two wings are of different length.
  • the number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more for a core. In some embodiments, the number is about 5-15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, for a core. In some embodiments, the number is 1 for a core. In some embodiments, the number is 2 for a core. In some embodiments, the number is 3 for a core. In some embodiments, the number is 4 for a core. In some embodiments, the number is 5 for a core. In some embodiments, the number is 6 for a core. In some embodiments, the number is 7 for a core. In some embodiments, the number is 8 for a core.
  • the number is 9 for a core. In some embodiments, the number is 10 for a core. In some embodiments, the number is 11 for a core. In some embodiments, the number is 12 for a core. In some embodiments, the number is 13 for a core. In some embodiments, the number is 14 for a core. In some embodiments, the number is 15 for a core.
  • a wing-core-wing is described as "X-Y-Z", where "X” represents the length of the 5' wing (as number of nucleobases), “Y” represents the length of the core (as number of nucleobases), and “Z” represents the length of the 3' wing (as number of nucleobases).
  • Example embodiments of X, Y, and Z include those lengths described as numbers (e.g., above) and exemplified in oligonucleotide species (e.g., in Table 1).
  • the two wings are of the same or different lengths and/or have the same or different modifications or patterns of modifications.
  • Y is between 8 and 15.
  • X, Y or Z can each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, each of X, Y and Z is independently 1-30. In some embodiments, X-Z-Z is 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5, 5-8-4, 4-8- 5, 5-7- 5, 4-7-5, 5-7-4, or 4-7-4. [00120] In some embodiments, a wing comprises one or more sugar modifications. In some embodiments, each sugar in a wing is independently modified.
  • each wing sugar in an oligonucleotide is independently modified.
  • each modified sugar independently comprises a 2’- modification (e.g., a 2’-OR s modified sugar, a LNA sugar, etc.).
  • each wing sugar is independently a 2’-OR s modified sugar.
  • each sugar modification in a wing is the same.
  • a wing comprises different sugar modifications, e.g., different 2’-OR s modifications.
  • 2’-OR s is 2’-OMe.
  • 2’-OR s is 2’-MOE.
  • each sugar in a wing is a 2’-MOE modified sugar.
  • each sugar in a wing is a 2’-OMe modified sugar.
  • a wing comprises one or more 2’-OMe modified sugars and one or more 2’-MOE modified sugars.
  • the two wings of a wing-core-wing structure comprise different sugar modifications or patterns thereof.
  • certain sugar modifications, e.g., 2’-MOE provide more stability under certain conditions than other sugar modifications, e.g., 2’-OMe or natural DNA or RNA sugars.
  • a wing comprises a bicyclic sugar.
  • a bicyclic sugar is a LNA, a cEt or a BNA sugar.
  • one or more internucleotidic linkages bonded to a 5’-wing sugar are each independently a modified internucleotidic linkage. In some embodiments, they are each independently a phosphorothioate internucleotidic linkage. In some embodiments, each internucleotidic linkage bonded to a 5’-wing sugar is independently a modified internucleotidic linkage. In some embodiments, each such internucleotidic linkage is independently a phosphorothioate internucleotidic linkage.
  • one or more internucleotidic linkages bonded to a 3’-wing sugar are each independently a modified internucleotidic linkage. In some embodiments, they are each independently a phosphorothioate internucleotidic linkage. In some embodiments, each internucleotidic linkage bonded to a 3’-wing sugar is independently a modified internucleotidic linkage. In some embodiments, each such internucleotidic linkage is independently a phosphorothioate internucleotidic linkage.
  • a core comprises one or more, e.g., about 1-20, 5-20, 6-20, 7-20, 8-20, 9- 20, 10-20, or 5-15, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 natural DNA sugars.
  • a core comprises 2 or more natural DNA sugars.
  • a core comprises 3 or more natural DNA sugars.
  • a core comprises 4 or more natural DNA sugars.
  • a core comprises 5 or more natural DNA sugars.
  • a core comprises 6 or more natural DNA sugars.
  • a core comprises 7 or more natural DNA sugars.
  • a core comprises 8 or more natural DNA sugars. In some embodiments, a core comprises 9 or more natural DNA sugars. In some embodiments, a core comprises 10 or more natural DNA sugars. In some embodiments, a core comprises 11 or more natural DNA sugars. In some embodiments, a core comprises 12 or more natural DNA sugars. In some embodiments, a core comprises 13 or more natural DNA sugars. In some embodiments, a core comprises 14 or more natural DNA sugars. In some embodiments, a core comprises 15 or more natural DNA sugars. In some embodiments, such DNA sugars are consecutive. In some embodiments, each sugar in a core is independently a natural DNA sugar.
  • one or more internucleotidic linkages bonded to a core sugar are each independently a modified internucleotidic linkage. In some embodiments, they are each independently a phosphorothioate internucleotidic linkage. In some embodiments, each internucleotidic linkage bonded to a core sugar is independently a modified internucleotidic linkage. In some embodiments, each such internucleotidic linkage is independently a phosphorothioate internucleotidic linkage.
  • a core is able to hybridize to a target mRNA, forming a duplex structure recognizable by RNase H, such that RNase H is able to cleave the mRNA.
  • Oligonucleotides [00129] Among other things, the present disclosure provides various oligonucleotides. As described herein, oligonucleotides may contain various nucleobase modifications, sugar modifications, internucleotidic linkages and patterns thereof. In some embodiments, the present disclosure provides the oligonucleotides in Table 1 as examples.
  • an internucleotidic linkage is a natural phosphate linkage.
  • each of A, T, C and G is independently deoxyadenosine, thymidine, deoxycytidine, and deoxyguanosine, respectively (e.g., as typically found in natural DNA).
  • “2MOEr” indicates a 2’-MOE modification to a sugar
  • “5” indicates a nucleoside has a 5’-OH group (e.g., when at the 5’-end of an oligonucleotide)
  • “3” indicates a nucleoside has a 3’-OH group (e.g., when at the 3’-end of an oligonucleotide)
  • “i” indicates an nucleoside is in the middle of an oligonucleotide and its 5’- and 3’- positions are bonded to internucleotidic linkages as indicated
  • “Me-dC” indicates a 5-methyl-2’- deoxycytidine nucleoside.
  • oligonucleotides may exist in various forms including various salt forms.
  • provided oligonucleotides are capable of hybridizing to a calpain-2 transcript. In some embodiments, provided oligonucleotides can reduce levels of calpain-2 transcripts or products thereof. In some embodiments, provided oligonucleotide can reduce levels of calpain-2 mRNA. In some embodiments, provided oligonucleotide can reduce levels of calpain-2 polypeptides. In some embodiments, provided oligonucleotide can reduce activity levels of calpain-2 polypeptides observed in a system (e.g., a sample, a subject, etc.). In some embodiments, an oligonucleotide is selected from Table 1.
  • an oligonucleotide is a pharmaceutically acceptable salt of an oligonucleotide selected from Table 1.
  • the present disclosure provides oligonucleotides that are particularly effective in reducing levels of calpain-2 transcripts, polypeptides and/or activities.
  • an oligonucleotide has the structure of /52MOErA/*/i2MOErT/*/i2MOErC/*/i2MOErA/*/i2MOErG/*T*T*T*/iMe- dC/*T*G*T*A*G*G*/i2MOErC/*/i2MOErT/*/i2MOErT/*/32MOErC/ or a salt thereof.
  • an oligonucleotide has the structure of /52MOErG/*/i2MOErG/*/i2MOErC/*/i2MOErA/*/i2MOErT/*A*/iMe-dC/*T*G*G*T*T*/iMe- dC/*A*G*/i2MOErT/*/i2MOErT/*/i2MOErG/*/i2MOErA/*/32MOErT/ or a salt thereof.
  • an oligonucleotide has the structure of /52MOErG/*/i2MOErC/*/i2MOErT/*/i2MOErC/*/i2MOErA/*G*G*T*/iMe-dC/*A*G*G*/iMe- dC/*A*G*/i2MOErT/*/i2MOErG/*/i2MOErT/*/32MOErT/ or a salt thereof.
  • an oligonucleotide has the structure of /52MOErG/*/i2MOErA/*/i2MOErG/*/i2MOErA/*/i2MOErG/*/iMe-dC/*/iMe- dC/*T*T*T*T*G*/iMe-dC/*A*/i2MOErG/*/i2MOErA/*/i2MOErG/*/i2MOErC/*/32MOErT/ or a salt thereof.
  • an oligonucleotide has the structure of /52MOErT/*/i2MOErC/*/i2MOErC/*/i2MOErA/*/i2MOErG/*/iMe-dC/*T*/iMe-dC/*T*G*T*G*/iMe- dC/*/iMe-dC/*T*/i2MOErC/*/i2MOErT/*/i2MOErA/*/i2MOErG/*/32MOErT/ or a salt thereof.
  • the present disclosure provides compositions comprising provided oligonucleotides.
  • an oligonucleotide composition comprises a provided oligonucleotide or a salt thereof, and various diastereomers and salt thereof. In some embodiments, an oligonucleotide composition comprises a provided oligonucleotide or a salt thereof, and various diastereomers with respect to chiral linkage phosphorus and salt thereof. In some embodiments, oligonucleotides may exist in one or more forms. In some embodiments, oligonucleotides in a composition exist in a salt form. In some embodiments, oligonucleotides in a composition exist in one or more salt forms. In some embodiments, a salt form is a pharmaceutically acceptable salt form.
  • a salt form is a metal salt. In some embodiments, a salt form is a alkali metal salt. In some embodiments, a salt form is a sodium salt. In some embodiments, a salt form is a potassium salt. In some embodiments, a salt form is a calcium salt. In some embodiments, a salt form is an ammonium salt form (e.g., of N(R’) 3 wherein R’ is as described herein; in some embodiments, each R’ is independently ⁇ H or optionally substituted C 1-6 alkyl). In some embodiments, an oligonucleotide composition is a liquid composition, wherein an oligonucleotide is dissolved in a solution.
  • a solution is a buffer. In some embodiments, a solution is a buffered saline. In some embodiments, in a composition acidic internucleotidic linkages, e.g., natural phosphate linkages, phosphorothioate internucleotidic linkages, e.g., independently exist as anionic forms, and the composition comprises one or more types of cations, e.g., Na + , K + , etc. Additional Chemical Moieties [00138] In some embodiments, an oligonucleotide comprises one or more additional chemical moieties. Various additional chemical moieties, e.g., targeting moieties, carbohydrate moieties, lipid moieties, etc.
  • certain additional chemical moieties facilitate delivery of oligonucleotides to desired cells, tissues and/or organs, including but not limited the cells of the central nervous system.
  • certain additional chemical moieties facilitate internalization of oligonucleotides.
  • certain additional chemical moieties increase oligonucleotide stability.
  • the present disclosure provides technologies for incorporating various additional chemical moieties into oligonucleotides.
  • oligonucleotides are manufactured on solid support using phosphoramidites.
  • oligonucleotides are manufactured in solution.
  • manufacturing of oligonucleotides comprise multiple cycles, in each of which one or more nucleoside units, typically one, are added.
  • a cycle comprises coupling of a phosphoramidite, blocking unreacted 5’-OH groups, modification (e.g., sulfurization), and/or de-blocking protected 5’-OH groups in newly coupled nucleosides.
  • modification may be performed at the end of cycles when certain lengths of oligonucleotides are achieved.
  • Certain technologies for manufacturing oligonucleotides are described in US 3687808, US 4469863, US 4476301, US 5177195, US 5023243, US 5034506, US 5166315, US 5185444, US 5188897, US 5214134, US 5216141, US 5235033, US 5264423, US 5264564, US 5276019, US 5278302, US 5286717, US 5321131, US 5399676, US 5405938, US 5405939, US 5434257, US 5453496, US 5455233, US 5466677, US 5466677, US 5470967, US 5476925, US 5489677, US 5519126, US 5536821, US 5541307, US 5541316, US 5550111, US 5561225, US 5563253, US 5571799, US 5587361, US 55
  • oligonucleotides and/or compositions are provided as stereorandom compositions with respect to chiral linkage phosphorus.
  • chiral linkages may be formed with no or low stereoselectivity.
  • oligonucleotides are provided as a mixture of various diastereomers and/or salts thereof.
  • a composition comprises an oligonucleotide, and/or one or more or all of its diastereomers with respect to chiral linkage phosphorus.
  • an oligonucleotide and/or its diastereomers are independently in one or more forms. In some embodiments, an oligonucleotide and/or its diastereomers are independently in one or more salt forms, e.g., one or more pharmaceutically acceptable salt forms. In some embodiments, for each chiral linkage phosphorus, both the Rp and the Sp configurations are present in the composition. In some embodiments, for each chiral linkage phosphorus, both the Rp and the Sp configurations have a percentage of at least about 10%, 15%, 20%, 25%, 30% 35%, 40%, or 45%.
  • both the Rp and the Sp configurations have a percentage of about 50%. In some embodiments, for each chiral internucleotidic linkage both the Rp and the Sp configurations have a percentage of about 50%. In some embodiments, for each chiral internucleotidic linkage both the Rp and the Sp configurations have a percentage of about 20-80%. In some embodiments, for each chiral internucleotidic linkage both the Rp and the Sp configurations have a percentage of about 30-70%. In some embodiments, for each chiral internucleotidic linkage both the Rp and the Sp configurations have a percentage of about 40-60%.
  • both the Rp and the Sp configurations have a percentage of about 45-55%.
  • the Rp configuration independently has a percentage of about 20-80%, 30-70%, 40-60%, or 45-55%, or about 20%, 30%, 40%, 50%, 60%, 70% or 80%.
  • Amount, concentration, etc., of provided oligonucleotides may be assessed utilizing various technologies in accordance with the present disclosure, e.g., by UV (e.g., at 260 nm), weight, etc. In some embodiments, amount, concentration, etc., of all oligonucleotides present in a composition is assessed.
  • calpain-2 refers to a gene or a gene product thereof (e.g., a nucleic acid (e.g., DNA, RNA, etc.), a transcript (e.g., a calpain-2 mRNA), a protein encoded thereby (e.g., a calpain-2 polypeptide), etc.) from a species, which may be known as CAPN2, CANP2, CANPL2, mCANP, CANPml, calpain-2, calpain 2, m-calpain, millimolar-calpain, calpain M-type, calpain-2 large subunit, calpain 2 (m/ll) large subunit,
  • CAPN2 sequences including variants thereof are readily available to those of skill in the art.
  • Various technologies e.g., assays, cells, animal models, etc., have also been reported and can be utilized for characterization and/or assessment of provided technologies (e.g., oligonucleotides, compositions, methods, etc.) in accordance with the present disclosure.
  • the CAPN2 gene is reported to encode a calpain-2 protein, which according to various reports comprises 622 or 700 amino acids, depending on isoform, and primarily localizes to cellular cytoplasm and cellular plasma membrane in a variety of tissues including those of a central nervous system (CNS).
  • CNS central nervous system
  • human calpain-2 comprises multiple domains including, from N- terminal region to C-terminal region: (i) an alpha helix; (ii) a CysPc domain comprising a first protease core domain (PC1) and a second protease core domain (PC2); (iii) a calpain-type beta-sandwich (CBSW) domain; and (iv) a penta-EF-hand in the catalytic large subunit (PEF(L)) domain.
  • Calpain-2 proteins from other species e.g., monkeys, rats, and mice have been reported to comprise various conserved domains as human calpain-2.
  • Calpain-2 reportedly belongs to a family of calcium-dependent proteases, and has been reported to cleave a multitude of protein targets including, e.g., actin, cytoplasmic polyadenylation element- binding protein 3 (CPEB3), p35, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), protein tyrosine phosphatase (PTPN13; also known as Fas-associated protein-1 (FAP1)), spectrin, TDP-43, neurofilament-light chain (Wang et al., Expert Opin. Ther. Targets, 2018; Baudry, Curr. Neuropharmacol., 2019), etc.
  • actin cytoplasmic polyadenylation element- binding protein 3 (CPEB3), p35, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), protein tyrosine phosphatase (PTPN13; also known as Fas-associated protein-1 (FAP1)),
  • calpain-2 Activation of calpain-2 has been reported to involve relatively high concentrations of calcium ion (Ca 2+ ), with reported ranges comprising near-millimolar amounts, e.g., 400-800 ⁇ M.
  • calpain-2 may be capable of being activated by phosphorylation by epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) via extracellular signal-regulated kinase (ERK) (Glading et al., Mol. Cell Biol., 2004; Zadran et al., J. Neurosci., 2010).
  • EGF epidermal growth factor
  • BDNF brain-derived neurotrophic factor
  • ERK extracellular signal-regulated kinase
  • Calpain-2 has been reported as a regulator of synaptic plasticity and possibly limiting such plasticity (Baudry and Bi, Trends Neurosci., 2017), and has been reported to be involved in excitotoxicity as inhibition of calpain-2 has reduced such toxicity (Wang et al., J. Neurosci., 2013). [00147] Dysregulation of calpain-2 has been reported to be associated with various forms of neurodegeneration. Calpain-2 has been implicated in neurodegenerative conditions, disorders or diseases including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), etc.
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer’s disease
  • calpain- 2 inhibition may result in improvements in the localization of TDP-43 in TBI; TDP-43 has been reported to form inclusions in some forms of neurodegeneration (Gao et al., J. Neurochem., 2018). As such, calpain- 2 may be a potential therapeutic target for both progressive neurodegenerative diseases as well as acute neuronal injury.
  • CAPN2-Associated Conditions, Disorders or Diseases [00148] Various conditions, disorders or diseases are reported to be associated with calpain-2 and may be prevented or treated with present disclosure.
  • a disease, disorder, or condition is associated with calpain-2 if the presence, level, activity, and/or form of calpain-2 and/or products (e.g., transcripts, encoded proteins, etc.) thereof correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population).
  • a condition, disorder or disease associated with calpain-2 may be treated and/or prevented by reducing expression, level and/or activity of calpain-2 transcripts and/or proteins.
  • the present disclosure provides technologies for preventing and/or treating various conditions, disorders or diseases.
  • a condition, disorder, or disease is a neurodegenerative disease.
  • a condition, disorder or disease is amyotrophic lateral sclerosis (ALS).
  • a condition, disorder or disease is traumatic brain injury (TBI).
  • TBI traumatic brain injury
  • a condition, disorder or disease is Alzheimer’s disease (AD).
  • a condition, disorder or disease is Parkinson’s disease (PD).
  • PD Parkinson’s disease
  • FTD frontotemporal dementia
  • a condition, disorder or disease is progressive supranuclear palsy (PSP).
  • a condition, disorder or disease is corticobasal degeneration (CBD).
  • a condition, disorder or disease is Wolfram syndrome (WS).
  • a condition, disorder or disease is Friedreich’s ataxia (FRDA).
  • a condition, disorder or disease is multiple system atrophy (MSA).
  • a condition, disorder or disease is spinocerebellar ataxia (SCA).
  • a condition, disorder or disease is spinal muscular atrophy (SMA).
  • a condition, disorder or disease is Pick’s disease (PD).
  • a condition, disorder or disease is progressive motor atrophy.
  • a condition, disorder or disease is concussion.
  • a condition, disorder or disease is spinal cord injury (SCI).
  • a condition, disorder or disease is chronic traumatic encephalopathy (CTE).
  • a condition, disorder or disease is seizure. In some embodiments, a condition, disorder or disease is stroke. In some embodiments, a condition, disorder or disease is intracerebral hemorrhage. In some embodiments, a condition, disorder or disease is a tauopathy. In some embodiments, a condition, disorder or disease is associated with Wallerian degeneration. In some embodiments, a condition, disorder or disease is acute glaucoma. In some embodiments, a condition, disorder or disease is cancer. In some embodiments, a condition, disorder or disease is diabetes. In some embodiments, a condition, disorder or disease is chemotherapy-induced peripheral neuropathy.
  • properties and/or activities of provided oligonucleotides and compositions thereof can be characterized and/or assessed using various technologies available to those skilled in the art, e.g., biochemical assays (e.g., RNase H assays), cell based assays, animal models, clinical trials, etc. Certain useful technologies are described in the Examples. Those skilled in the art reading the present disclosure will readily appreciate that other technologies, e.g., in vitro models (e.g., cell lines) for various conditions, disorders or diseases, animal models for various conditions, disorders or diseases, clinical trials, etc.
  • oligonucleotides are useful for many purposes.
  • provided technologies e.g., oligonucleotides, compositions, methods, etc.
  • provided technologies reduce levels and/or activities calpain-2 RNA transcripts.
  • provided oligonucleotides and compositions provide knockdown of calpain-2 mRNA.
  • provided technologies reduce levels of calpain-2 polypeptides.
  • provided technologies reduce levels of calpain-2 activities, e.g., protease activities.
  • the present disclosure provides a method for reducing level of calpain- 2 mRNA in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or an oligonucleotide composition.
  • the present disclosure provides a method for reducing level of a calpain-2 polypeptide in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or an oligonucleotide composition. In some embodiments, the present disclosure provides a method for reducing level of a calpain-2 activity in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or an oligonucleotide composition.
  • the present disclosure provides a method for reducing level of calpain-2 protease activity in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or an oligonucleotide composition.
  • reduction of calpain-2 mRNA and/or polypeptide levels increases mRNA and/or polypeptide levels of a calpain-2 target.
  • reduction of calpain-2 mRNA and/or polypeptide levels increases mRNA and/or polypeptide levels of calpastatin.
  • the present disclosure provides technologies for increasing levels of calpastatin mRNA and/or polypeptide in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or an oligonucleotide composition that targets calpain-2.
  • Calpain-2 has been reported to interact with various partners, e.g., TDP43 and other ALS biomarkers.
  • the present disclosure provide technologies for modulating interaction between calpain-2 and a partner.
  • a system comprises calpain-2 mRNA.
  • a system expresses calpain-2 mRNA.
  • a system expresses calpain-2 polypeptides.
  • a system is an in vitro system.
  • a system is an in vivo system.
  • a system comprises a cell.
  • a system is a cell. In some embodiments, a system comprises a population of cells. In some embodiments, a system is a population of cells. In some embodiments, a cell is a neuronal cell. In some embodiments, a cell is a cell in the neuronal system. In some embodiments, a cell is a cell in CNS. In some embodiments, a cell possesses one or more characteristics, properties and/or activities of a neuronal cell. [00156] In some embodiments, a system is a tissue. In some embodiments, a system comprises a tissue. In some embodiments, a system is an organ. In some embodiments, a system comprises an organ.
  • a system is a brain or a portion thereof. In some embodiments, a system comprises a brain or a portion thereof. In some embodiments, a system is an organism. In some embodiments, a system comprises an organism. In some embodiments, a system is a subject. In some embodiments, a system is a mammal, e.g., a mouse, rat, monkey, etc. In some embodiments, a system is a human.
  • a level is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% compared to absence of a provided oligonucleotide or composition and/or presence of a reference oligonucleotide or composition. In some embodiments, such a reduction is achieved at certain oligonucleotide concentrations (e.g., 1 nM, 5 nM, 10 nM, 100 nM, 500 nM, 1 uM, 5 uM, etc.) or doses.
  • oligonucleotide concentrations e.g., 1 nM, 5 nM, 10 nM, 100 nM, 500 nM, 1 uM, 5 uM, etc.
  • a reference composition comprises no oligonucleotides targeting calpain-2.
  • a reference oligonucleotide targets a different nucleic acid than calpain-2.
  • a level is of mRNA, e.g., calpain 2 mRNA.
  • a level is of a polypeptide, e.g., calpain 2 protein.
  • a level is reduced by at least about 10%.
  • a level is reduced by at least about 20%. In some embodiments, a level is reduced by at least about 30%. In some embodiments, a level is reduced by at least about 40%. In some embodiments, a level is reduced by at least about 50%. In some embodiments, a level is reduced by at least about 60%. In some embodiments, a level is reduced by at least about 70%. In some embodiments, a level is reduced by at least about 75%. In some embodiments, a level is reduced by at least about 80%. In some embodiments, a level is reduced by at least about 85%. In some embodiments, a level is reduced by at least about 90%. In some embodiments, a level is reduced by at least about 95%.
  • level of calpain-2 mRNA is reduced about or at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% at about 20 uM oligonucleotides when assessed, e.g., using an assay in the Examples; in some embodiments, it is reduced about or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% at about 15 uM oligonucleotides; in some embodiments, it is reduced about or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% at about 10 uM oligonucleotides; in some embodiments, it is reduced about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% at about 5 uM oligonucleotides; in some embodiments
  • a reduction is about or at least about 50%. In some embodiments, a reduction is about or at least about 55%. In some embodiments, a reduction is about or at least about 60%. In some embodiments, a reduction is about or at least about 65%. In some embodiments, a reduction is about or at least about 70%. In some embodiments, a reduction is about or at least about 75%. In some embodiments, a reduction is about or at least about 80%. In some embodiments, a reduction is about or at least about 85%. In some embodiments, a reduction is about or at least about 90%. In some embodiments, a reduction is about or at least about 95%.
  • a reduction is assessed at or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, or about 1, 2, 3 or 4 weeks following administering or delivering a provided oligonucleotide or composition. In some embodiments, a reduction is assessed at or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, or about 1, 2, 3 or 4 weeks following removal or washout of a provided oligonucleotide or composition. In some embodiments, a reduction is assessed at day 0. In some embodiments, a reduction is assessed at about day 3. In some embodiments, a reduction is assessed at about day 10.
  • a reduction is assessed at about day 14. In some embodiments, a reduction is assessed at about day 21. In some embodiments, reductions of at one or more assessments, e.g., of calpain mRNA levels, are independently about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%; in some embodiments, reductions are independently about or at least about 75%; in some embodiments, reductions are about or at least about 80%.
  • reductions of one or more assessments are independently about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%; in some embodiments, reductions are about or at least about 20%; in some embodiments, reductions are about or at least about 25%; in some embodiments, reductions are about or at least about 30%; in some embodiments, reductions are about or at least about 35%; in some embodiments, reductions are about or at least about 40%; in some embodiments, reductions are about or at least about 45%; in some embodiments, reductions are about or at least about 50%; in some embodiments, reductions are about or at least about 55%; in some embodiments, reductions are about or at least about 60%; in some embodiments, reductions are about or at least about 70%.
  • reductions are relative to absence of oligonucleotides. In some embodiments, reductions are relative to presence of reference oligonucleotides or compositions, e.g., oligonucleotides targeting TUG1 as described herein. In some embodiments, assessments are performed according to those described in the Examples, e.g., in some embodiments, using iPSC-derived motor neurons with oligonucleotide concentrations at about 20 uM with gymnotic delivery.
  • activity of a provided oligonucleotide or oligonucleotide composition may be assessed by IC50 which is the concentration to reduce a level, e.g., of calpain-2 mRNA, polypeptide, activity, etc., by 50% in a suitable condition, e.g., cell-based in vitro assays, assays described in the Examples, etc.
  • IC50 is the concentration to reduce a level, e.g., of calpain-2 mRNA, polypeptide, activity, etc.
  • a suitable condition e.g., cell-based in vitro assays, assays described in the Examples, etc.
  • provided oligonucleotides or compositions have an IC50 of about or no more than about 0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 5000 or 10000 nM.
  • an oligonucleotide has an IC50 of about or no more than about 10000 nM. In some embodiments, an oligonucleotide has an IC50 of about or no more than about 5000 nM. In some embodiments, an oligonucleotide has an IC50 of about or no more than about 2000 nM. In some embodiments, an oligonucleotide has an IC50 of about or no more than about 1000 nM. In some embodiments, an IC50 is about or no more than about 500 nM. In some embodiments, an IC50 is about or no more than about 200 nM. In some embodiments, an IC50 is about or no more than about 100 nM.
  • an IC50 is about or no more than about 50 nM. In some embodiments, an IC50 is about or no more than about 20 nM. In some embodiments, an IC50 is about or no more than about 10 nM. In some embodiments, an IC50 is about or no more than about 5 nM. In some embodiments, an IC50 is about or no more than about 2 nM. In some embodiments, an IC50 is about or no more than about 1 nM.
  • provided oligonucleotides and compositions are useful for treating various conditions, disorders or diseases, by reducing levels and/or activities of calpain-2 transcripts and/or products encoded thereby that are associated with the conditions, disorders or diseases.
  • the present disclosure provides method for preventing a condition, disorder or disease, comprising administering or delivering to a subject susceptible thereto an effective amount of an oligonucleotide or composition of the present disclosure.
  • the present disclosure provides method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide or composition of the present disclosure.
  • a condition, disorder or disease is a neurodegenerative condition, disorder or disease.
  • a condition, disorder or disease is or comprises Wallerian degeneration.
  • Wallerian degeneration is associated with damage.
  • Wallerian degeneration is associated with stress.
  • a condition, disorder or disease is associated with Wallerian degeneration.
  • a condition, disorder or disease is amyotrophic lateral sclerosis (ALS).
  • a condition, disorder or disease is neuropathy.
  • a condition, disorder or disease is peripheral neuropathy.
  • a condition, disorder or disease is peripheral neuropathy induced by chemotherapy.
  • a condition, disorder or disease is Parkinson’s disease.
  • a condition, disorder or disease is Huntington’s disease.
  • a condition, disorder or disease is Alzheimer’s disease.
  • a condition, disorder or disease is frontotemporal dementia.
  • a condition, disorder or disease is brain injury.
  • a condition, disorder or disease is traumatic brain injury.
  • a condition, disorder or disease is progressive supranuclear palsy.
  • a condition, disorder or disease is corticobasal degeneration.
  • a condition, disorder or disease is Wolfram Syndrome.
  • a condition, disorder or disease is Friedreich's Ataxia.
  • a condition, disorder or disease is Multiple System Atrophy.
  • a condition, disorder or disease is Spinal Cerebellar Ataxia.
  • a condition, disorder or disease is Spinal Muscular Atrophy (SMA).
  • SMA Spinal Muscular Atrophy
  • a condition, disorder or disease is Pick's Disease.
  • a condition, disorder or disease is progressive motor atrophy.
  • a condition, disorder or disease is associated with neuronal damage. In some embodiments, a condition, disorder or disease is associated with neuronal cell damage. In some embodiments, a condition, disorder or disease is associated with neuronal cell death. In some embodiments, a condition, disorder or disease is axonal degeneration. In some embodiments, a condition, disorder or disease is axonal degeneration is ALS. [00163] Various technologies may be utilized to administer or deliver provided oligonucleotides or compositions. In some embodiments, an oligonucleotide or a composition is administered or delivered orally. In some embodiments, an oligonucleotide or a composition is administered or delivered via parenteral routes.
  • parenteral routes include intravenous, intrarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes.
  • an oligonucleotide or a composition is administered or delivered via intraocular, intraorbital, subconjuctival, intravitreal, subretinal, transscleral or introcochlear route.
  • an oligonucleotide or a composition is administered or delivered parenterally.
  • an oligonucleotide or a composition is administered or delivered intrathecally.
  • an oligonucleotide or a composition is administered or delivered intravenously.
  • oligonucleotides are administered or delivered as a liquid composition. In some embodiments, oligonucleotides are dissolved in a liquid, e.g., a buffered saline such as aCSF, for administration or delivery.
  • a liquid e.g., a buffered saline such as aCSF
  • an oligonucleotide or composition may be utilized in combination with another therapy, e.g., another therapeutic agent.
  • provided technologies e.g., oligonucleotides, compositions, methods, etc., delay or prevent onset of or more symptoms and/or hallmarks of a condition, disorder or disease. In some embodiments, provided technologies delay, slow down, or prevent progression of a condition, disorder or disease.
  • provided technologies alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • provided technologies improve performance of a subject in one or more assessments.
  • provided technologies improve performance of a subject in one or more clinical assessments.
  • provided technologies independently improve one or more clinical assessment results of a subject.
  • Pharmaceutical Compositions comprising a provided compound, e.g., an oligonucleotide, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier.
  • oligonucleotides of the present disclosure are provided as pharmaceutical compositions.
  • oligonucleotides can be provided in various forms.
  • oligonucleotides can be in acid forms, e.g., for natural phosphate linkages, in the form of ⁇ OP(O)(OH)O ⁇ ; for phosphorothioate internucleotidic linkages, in the form of ⁇ OP(O)(SH)O ⁇ ; etc.
  • provided oligonucleotides can be in salt forms, e.g., for natural phosphate linkages, in the form of ⁇ OP(O)(ONa)O ⁇ in sodium salts; for phosphorothioate internucleotidic linkages, in the form of ⁇ OP(O)(SNa)O ⁇ in sodium salts; etc.
  • oligonucleotides of the present disclosure can exist in acid, base and/or salt forms.
  • a composition comprises one or more forms of an oligonucleotide.
  • a composition comprises one or more salt forms of an oligonucleotide.
  • a composition comprises one or more pharmaceutically acceptable salt forms of an oligonucleotide.
  • a provided oligonucleotide or composition is typically administered as a pharmaceutical composition.
  • a pharmaceutical composition is suitable for administration or delivery of an oligonucleotide to an area or portion of a body affected by a condition, disorder or disease.
  • a pharmaceutical composition comprises a therapeutically effective amount of a provided oligonucleotide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises a therapeutically effective amount of oligonucleotides which are diastereomers of each other, wherein the oligonucleotides exist in one or more forms. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of oligonucleotides which are diastereomers of each other with respect to chiral linkage phosphorus, wherein the oligonucleotides exist in one or more forms. [00169] In some embodiments, a pharmaceutically acceptable carrier is a buffer. In some embodiments, a pharmaceutically acceptable carrier is a buffered saline. In some embodiments, a pharmaceutically acceptable carrier is artificial cerebrospinal fluid.
  • a composition is a liquid composition comprising dissolved oligonucleotides.
  • a pharmaceutical composition is formulated for intravenous injection, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration or otic administration.
  • a pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.
  • a pharmaceutical composition is formulated for intrathecal administration.
  • oligonucleotides may exist in various salt forms.
  • a salt is a pharmaceutically acceptable salt.
  • a pharmaceutical composition comprises an oligonucleotide, optionally in its salt form, and a sodium salt.
  • a pharmaceutical composition comprises an oligonucleotide, optionally in its salt form, and sodium chloride.
  • each hydrogen ion of an oligonucleotide that may be donated to a base is replaced by a non-H + cation.
  • a pharmaceutically acceptable salt of an oligonucleotide is an all-metal ion salt, wherein each hydrogen ion (for example, of ⁇ OH, ⁇ SH, etc.) of each internucleotidic linkage (e.g., a natural phosphate linkage, a phosphorothioate internucleotidic linkage, etc.) is replaced by a metal ion.
  • a pharmaceutically acceptable salt is a sodium salt.
  • a pharmaceutically acceptable salt is magnesium salt.
  • a pharmaceutically acceptable salt is a calcium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is an ammonium salt (cation N(R’) 4 + ). In some embodiments, a pharmaceutically acceptable salt comprises one and no more than one types of cation. In some embodiments, a pharmaceutically acceptable salt comprises two or more types of cation. In some embodiments, a cation is Li + , Na + , K + , Mg 2+ or Ca 2+ . In some embodiments, a pharmaceutically acceptable salt is an all-sodium salt.
  • a pharmaceutically acceptable salt is an all-sodium salt, wherein each internucleotidic linkage which is a natural phosphate linkage (acid form ⁇ O ⁇ P(O)(OH) ⁇ O ⁇ ), if any, exists as its sodium salt form ( ⁇ O ⁇ P(O)(ONa) ⁇ O ⁇ ), and each internucleotidic linkage which is a phosphorothioate internucleotidic linkage (acid form ⁇ O ⁇ P(O)(SH) ⁇ O ⁇ ), if any, exists as its sodium salt form ( ⁇ O ⁇ P(O)(SNa) ⁇ O ⁇ ).
  • an oligonucleotide or a composition e.g., a pharmaceutical composition
  • an oligonucleotide or a composition is provided as solid.
  • an oligonucleotide or a composition e.g., a pharmaceutical composition
  • an oligonucleotide or a composition e.g., a pharmaceutical composition
  • Various technologies for delivering nucleic acids and/or oligonucleotides are known in the art and can be utilized in accordance with the present disclosure.
  • Example nanocarriers can be used to deliver nucleic acids.
  • Example nanocarriers include liposomes, cationic polymer complexes and various polymeric compounds. Complexation of nucleic acids with various polycations is another approach for intracellular delivery; this includes use of PEGylated polycations, polyethyleneamine (PEI) complexes, cationic block co-polymers, and dendrimers.
  • PEI polyethyleneamine
  • cationic block co-polymers cationic block co-polymers
  • dendrimers dendrimers.
  • PEI polyethyleneamine
  • Several cationic nanocarriers, including PEI and polyamidoamine dendrimers may help to release contents from endosomes.
  • oligonucleotide is conjugated to another molecule.
  • oligonucleotides are administered or delivered via gymnotic uptake.
  • oligonucleotides or compositions are formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington, The Science and Practice of Pharmacy (20th ed. 2000).
  • oligonucleotides or compositions are delivered to CNS. In certain embodiments, oligonucleotides and compositions are delivered to cerebrospinal fluid. In certain embodiments, oligonucleotides and compositions are administered to brain parenchyma.
  • oligonucleotides and compositions are delivered to an animal/subject by intrathecal administration, or intracerebroventricular administration. Broad distribution of oligonucleotides and compositions may be achieved with methods of administration described herein and/or known in the art.
  • parenteral administration is by injection, by, e.g., a syringe, a pump, etc.
  • an injection is a bolus injection.
  • an injection is administered directly to a tissue or location, such as cerebrospinal fluid, striatum, caudate, cortex, hippocampus and/or cerebellum.
  • oligonucleotides and compositions thereof are effective over a wide dose range.
  • a dose is from about 0.01 to about 1000 mg, from about 0.5 to about 100 mg, from about 1 to about 50 mg, or from about 5 to about 100 mg.
  • Exact doses may depend upon routes of administration, forms in which oligonucleotides are administered, subjects (e.g., body weight, age, body surface area, etc.), conditions, disorders or diseases, and/or preferences and experiences of physicians.
  • a fixed dose is administered.
  • a provided oligonucleotide or composition is administered or delivered, e.g., by injection or infusion, once every week, every two weeks, every month, every two months, every 90 days, every 3 months, every 6 months, every 9 months, or once a year.
  • two or more doses are about the same amount.
  • one or more doses are independently more than one or more other doses.
  • one or more loading doses each independently of a higher amount are administered before one or more maintenance doses each independently of a lower amount.
  • two or more or all loading doses are about the same amount.
  • a loading dose is of a higher amount than another loading dose.
  • Example Embodiments [00180] Among other things, the present disclosure provides the following Example Embodiments: 1. An oligonucleotide, wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of ATCAGTTTCTGTAGGCTTCC, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage. 2.
  • the oligonucleotide of Embodiment 3 wherein the base sequence of the oligonucleotide is GGCATACTGGTTCAGTTGAT. 5.
  • an oligonucleotide wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of GCTCAGGTCAGGCAGTGGTT, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage. 6.
  • the oligonucleotide of Embodiment 5 wherein the base sequence of the oligonucleotide is GCTCAGGTCAGGCAGTGGTT. 7.
  • an oligonucleotide wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of GAGAGCCTTTTTGCAGAGCT, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage.
  • the base sequence of the oligonucleotide is GAGAGCCTTTTTGCAGAGCT.
  • an oligonucleotide wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of TCCAGCTCTGTGCCTCTAGT, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage. 10.
  • an oligonucleotide wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of GTTCCAGCTTGGGCAGTTGT, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage. 12.
  • an oligonucleotide wherein: the base sequence of the oligonucleotide comprises 10 or more contiguous nucleobases of GGAAGCTTAGTCCTTGGCTG, wherein each T is optionally and independently replaced with U; and the oligonucleotide comprises a modified nucleobase, a modified sugar or a modified internucleotidic linkage. 14. The oligonucleotide of Embodiment 13, wherein the base sequence of the oligonucleotide is GGAAGCTTAGTCCTTGGCTG. 15.
  • oligonucleotide of any one of the preceding Embodiments wherein a sugar in a 5’-wing is a 2’-OR s modified sugar wherein R s is C 1-6 aliphatic.
  • a sugar in a 5’-wing is a 2’-MOE modified sugar.
  • a sugar in a 5’-wing is a bicyclic sugar.
  • the bicyclic sugar is a LNA sugar.
  • the bicyclic sugar is a cEt sugar.
  • each sugar in a 5’-wing is independently a 2’-MOE modified sugar.
  • 27. The oligonucleotide of any one of the preceding Embodiments, wherein there are about 8-15 nucleosides in the gap.
  • 28. The oligonucleotide of any one of the preceding Embodiments, wherein there are 10 nucleosides in the gap.
  • 29. The oligonucleotide of any one of the preceding Embodiments, wherein each sugar in the gap is independently a natural DNA sugar.
  • 30. The oligonucleotide of any one of the preceding Embodiments, wherein the gap contains no cytosine.
  • oligonucleotide of any one of the preceding Embodiments wherein the gap comprises one or more 5-methylcytosine.
  • 32. The oligonucleotide of any one of the preceding Embodiments, wherein there are about 3-10 nucleosides in the 3’-wing.
  • 33. The oligonucleotide of any one of the preceding Embodiments, wherein there are 5 nucleosides in the 3’-wing.
  • oligonucleotide of any one of the preceding Embodiments wherein a sugar in a 3’-wing is a bicyclic sugar.
  • a sugar in a 3’-wing is a bicyclic sugar.
  • the bicyclic sugar is a LNA sugar.
  • the bicyclic sugar is a cEt sugar.
  • each sugar in a 3’-wing is independently a 2’-MOE modified sugar.
  • 43. The oligonucleotide of any one of the preceding Embodiments, wherein the oligonucleotide comprises a modified internucleotidic linkage.
  • 44. The oligonucleotide of Embodiment 43, wherein the modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.
  • 45 The oligonucleotide of any one of the preceding Embodiments, wherein each internucleotidic linkage is independently a modified internucleotidic linkage.
  • each internucleotidic linkage is independently a phosphorothioate internucleotidic linkage.
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 48.
  • An oligonucleotide having the structure of: /52MOErG/*/i2MOErC/*/i2MOErT/*/i2MOErC/*/i2MOErA/*G*G*T*/iMe-dC/*A*G*G*/iMe- dC/*A*G*/i2MOErT/*/i2MOErG/*/i2MOErT/*/32MOErT/ or a salt thereof, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 50.
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 51.
  • An oligonucleotide having the structure of: /52MOErG/*/i2MOErT/*/i2MOErT/*/i2MOErC/*/i2MOErC/*A*G*/iMe- dC/*T*T*G*G*G*/iMe-dC/*A*/i 2MOErG/*/i2MOErT/*/i2MOErT/*/i2MOErG/*/32MOErT/ or a salt thereof, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 53.
  • oligonucleotide of any one of the preceding Embodiments wherein the oligonucleotide is a pharmaceutically acceptable salt.
  • oligonucleotide of any one of the preceding Embodiments wherein the oligonucleotide is a sodium salt.
  • a composition comprising an oligonucleotide of any one of the preceding Embodiments and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus. 57.
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErA/*/i2MOErT/*/i2MOErC/*/i2MOErA/*/i2MOErG/*T*T*T*/iMe- dC/*T*G*T*A*G*G*/i2MOErC/*/i2MOErT/*/i2MOErT/*/i2MOErC/*/32MOErC/, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 58.
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErG/*/i2MOErG/*/i2MOErC/*/i2MOErA/*/i2MOErT/*A*/iMe-dC/*T*G*G*T*T*/iMe- dC/*A*G*/i2MOErT/*/i2MOErT/*/i2MOErG/*/i2MOErA/*/32MOErT/, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 59.
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErG/*/i2MOErC/*/i2MOErT/*/i2MOErC/*/i2MOErA/*G*G*T*/iMe-dC/*A*G*G*/iMe- dC/*A*G*/i2MOErT/*/i2MOErG/*/i2MOErT/*/32MOErT/, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively;
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErT/*/i2MOErC/*/i2MOErC/*/i2MOErA/*/i2MOErG/*/iMe-dC/*T*/iMe- dC/*T*G*T*G*/iMe-dC/*/iMe- dC/*T*/i2MOErC/*/i2MOErA/*/i2MOErG/*/32MOErT/, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 61.
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErG/*/i2MOErT/*/i2MOErT/*/i2MOErC/*/i2MOErC/*A*G*/iMe- dC/*T*T*G*G*G*/iMe-dC/*A*/i2MOErG/*/i2MOErT/*/i2MOErT/*/i2MOErG/*/32MOErT/, wherein: each of A, T and G is independently deoxyadenosine, thymidine
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErG/*/i2MOErG/*/i2MOErA/*/i2MOErA/*/i2MOErG/*/iMe-dC/*T*T*A*G*T*/iMe- dC/*/iMe-dC/*T*T*/i2MOErG/*/i2MOErG/*/i2MOErC/*/i2MOErT/*/32MOErG/, wherein:
  • each of A, T and G is independently deoxyadenosine, thymidine, and deoxyguanosine, respectively; 63.
  • a composition comprising: an oligonucleotide or a salt thereof, and one or more diastereomers of the oligonucleotide with respect to chiral linkage phosphorus, or one or more salts of the diastereomers, wherein the oligonucleotide is /52MOErG/*/i2MOErA/*/i2MOErG/*/i2MOErA/*/i2MOErG/*/iMe-dC/*/iMe- dC/*T*T*T*T*T*G*/iMe- dC/*A*/i2MOErG/*/i2MOErA/*/i2MOErG/*/i2MOErC/*/32MOErT/, wherein: each of A, T and G is independently deoxyadeno
  • composition of any one of Embodiments 56-63, wherein for each chiral linkage phosphorus, the percentage of the Rp configuration is independently about 20%-80%.
  • the composition of any one of Embodiments 56-63, wherein for each chiral linkage phosphorus, the percentage of the Rp configuration is independently about 30%-70%.
  • the composition of any one of Embodiments 56-63, wherein for each chiral linkage phosphorus, the percentage of the Rp configuration is independently about 40%-60%.
  • the composition of any one of Embodiments 56-63, wherein for each chiral linkage phosphorus, the percentage of the Rp configuration is independently about 45%-55%. 68.
  • composition of any one of Embodiments 56-70 wherein the composition comprises a pharmaceutically acceptable salt of the oligonucleotide, one or more pharmaceutically acceptable salts of one or more diastereomers, and a pharmaceutically acceptable carrier.
  • 72. A pharmaceutical composition comprising an oligonucleotide of any one of the preceding Embodiments and a pharmaceutically acceptable carrier.
  • 73. The composition of Embodiment 72, wherein the composition comprises one or more pharmaceutically acceptable salts of an oligonucleotide.
  • composition is a liquid composition.
  • 75 The composition of any one of Embodiments 70-74, wherein a pharmaceutically acceptable carrier is a buffer.
  • composition of any one of Embodiments 70-74, wherein a pharmaceutically acceptable carrier is a buffered saline.
  • a pharmaceutically acceptable carrier is artificial cerebrospinal fluid.
  • a method for reducing level of calpain-2 mRNA in a system comprising administering or delivering to the system an effective amount of an oligonucleotide or composition of any one of the preceding Embodiments.
  • a method for reducing level of calpain-2 activity in a system comprising administering or delivering to the system an effective amount of an oligonucleotide or composition of any one of the preceding Embodiments.
  • the method of any one of Embodiments 78-81, wherein the system is or comprises a tissue.
  • the method of any one of Embodiments 109-128, wherein the reference oligonucleotide targets TUG1 or the reference composition comprises oligonucleotides targeting TUG1.
  • 131. The method of any one of Embodiments 89-130, wherein the reduction is assessed at or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, or about 1, 2, 3 or 4 weeks following administering or delivering the oligonucleotide or composition.
  • 132. The method of any one of Embodiments 89-131, wherein the reduction is assessed at about D3 following administering or delivering the oligonucleotide or composition.
  • 134 The method of any one of Embodiments 89-133, wherein the reduction is assessed at about D10 following administering or delivering the oligonucleotide or composition. 135.
  • 136 The method of any one of Embodiments 89-135, wherein the reduction is assessed at about D21 following administering or delivering the oligonucleotide or composition. 137.
  • a method for preventing or treating a condition, disorder or disease comprising administering or delivering to a subject susceptible thereto an effective amount of an oligonucleotide or composition of any one of Embodiments 1-77.
  • a method for treating a condition, disorder or disease comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide or composition of any one of Embodiments 1-77. 148.
  • Embodiment 147 wherein severity of a symptom of the condition, disorder or disease is reduced.
  • 149 The method of any one of Embodiments 147-148, wherein one or more clinical assessment results of the subject are independently improved.
  • 150 The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is associated with Wallerian degeneration.
  • 151 The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is a neurodegenerative condition, disorder or disease.
  • 152 The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is amyotrophic lateral sclerosis. 153.
  • the method of any one of Embodiments 144-149, wherein a condition, disorder or disease is peripheral neuropathy. 154. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is peripheral neuropathy induced by chemotherapy. 155. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is Parkinson’s disease. 156. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is Huntington’s disease. 157. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is Alzheimer’s disease. 158.
  • a condition, disorder or disease is frontotemporal dementia.
  • a condition, disorder or disease is traumatic brain injury.
  • a condition, disorder or disease is progressive supranuclear palsy.
  • a condition, disorder or disease is corticobasal degeneration.
  • a condition, disorder or disease is Wolfram Syndrome. 163.
  • the method of any one of Embodiments 144-149, wherein a condition, disorder or disease is Friedreich’s Ataxia. 164. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is multiple system atrophy. 165. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is spinal cerebellar ataxia. 166. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is spinal muscular atrophy. 167. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is Pick’s Disease. 168.
  • the method of any one of Embodiments 144-149, wherein a condition, disorder or disease is progressive motor atrophy. 169. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is stroke. 170. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is concussion. 171. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is intracerebral hemorrhage. 172. The method of any one of Embodiments 144-149, wherein a condition, disorder or disease is acute glaucoma. 173.
  • the method of any one of Embodiments 144-149, wherein a condition, disorder or disease is seizure. 174.
  • the method of any one of Embodiments 144-149, wherein a condition, disorder or disease is spinal cord injury. 175.
  • a method for reducing NF-L excretion comprising administering or delivering to a subject an effective amount of an oligonucleotide or composition of any one of Embodiments 1-77. 177.
  • a method for reducing neuritic degeneration comprising administering or delivering to a subject an effective amount of an oligonucleotide or composition of any one of Embodiments 1-77. 179.
  • 180. The method of any one of Embodiments 144-179, wherein the oligonucleotide or composition is administered or delivered intrathecally.
  • iPSC human induced pluripotent stem cell
  • io1001 human induced pluripotent stem cell-derived glutamatergic neurons
  • various oligonucleotide compositions were added to the cells at a concentration of 5 ⁇ M in 5% TE buffer.
  • Cells were incubated with the oligonucleotides for 48 hours to allow for gymnotic uptake and knockdown of CAPN2 expression. Following this incubation period, cells were lysed and RNA was collected (Thermo Fisher cat. # A25603).
  • RNA was used in real time RT-qPCR to quantify fold-change of CAPN2 expression.
  • various oligonucleotides provided in vitro knockdown of calpain-2 expression.
  • various oligonucleotides provided substantial in vitro knockdown of CAPN2 expression as compared to reference conditions.
  • Negative control is /52MOErC/*/i2MOErC/*/i2MOErT/*/i2MOErA/*/i2MOErT/*A*G*G*A*/iMe-dC/*T*A*T*/iMe- dC/*/iMe-dC/*/i2MOErA/*/i2MOErG/*/i2MOErG/*/i2MOErA/*/32MOErA/.
  • the present disclosure provides oligonucleotides and compositions having low or no cytotoxicity.
  • human iPSC-derived glutamatergic neurons (bit.bio ioGlutamatergic Neurons, Cat. No.: io1001) were seeded at 20,000 cells/well in a 96-well format. Following culturing of the cells for 4 days, various oligonucleotides were added to the cells at a concentration of 5 ⁇ M in 5% TE buffer. Cells were incubated with the oligonucleotides for 48 hours. Following this incubation period, cells underwent Hoechst staining (5 ⁇ g/mL).
  • the number of live, dead, and total (live + dead) cells were counted and the percentage of live cells was calculated. As shown in Figure 3, no significant change in the percentage of live cells were observed following treatment with various oligonucleotides as compared to vehicle treatment (TE buffer).
  • Positive control is ⁇ T*G*C*A*A*>G*T*C*T*G*A*C*G*C*C* ⁇ C*A*T*C*T> (targeting NEAT1)
  • negative control is ⁇ A*C*C*A*G*>T*G*C*A*T*T*C*A*T*T* ⁇ C*G*A*G*T>
  • all nucleosides are DNA nucleosides except those in ⁇ > which are 2’-MOE modified, and each * independently represents a phosphorothioate internucleotidic linkage. Staurosporine was utilized to induce toxicity in a separate well.
  • oligonucleotides and compositions can provide dose-dependent reduction of calpain- 2 expression.
  • the present disclosure demonstrate that various provided oligonucleotide and compositions can provide dose-dependent reduction of calpain-2 mRNA levels. Results from an assessment are presented below as an example.
  • Cells Human Bit.bio ioGlutamatergic neurons, seeded at 20,000 cells/well in 96-well format. Thawing, seeding and culturing according to supplier’s instructions (coating: 0.01% PLO and 26 ng/cm2 laminin).
  • Oligonucleotide treatment oligonucleotide compositions 14, 15, 36, 39, 71, 77 and TUG1 (negative control). Concentration range: 20 ⁇ M, 15 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.63 ⁇ M, 0.31 ⁇ M. Positive control (targeting NEAT1): oligonucleotide targeting NEAT1 (5 ⁇ M). Negative control: TUG1 (5 ⁇ M). Vehicle: 20% TE buffer without oligonucleotides (same TE buffer content for all conditions). Oligonucleotides were added to cells 4 days after seeding the cells (by gymnotic uptake, 48 hours incubation).
  • Treatments were in duplicates on separate plates, except for negative control TUG1 oligonucleotides.
  • Positive control ⁇ T*G*C*A*A*>G*T*C*T*G*A*C*G*C*C* ⁇ C*A*T*C*T> (targeting NEAT1);
  • negative control ⁇ A*C*C*A*G*>T*G*C*A*T*T*C*T* ⁇ C*G*A*G*T>; all nucleosides are DNA nucleosides except those in ⁇ > which are 2’-MOE modified, and each * independently represents a phosphorothioate internucleotidic linkage.
  • a strong separation of positive and negative control oligonucleotide was observed. There was no marked intra- or inter-plate variability between positive and negative controls. No plate positional effects were observed based on control sample performance (vehicle, positive and negative controls), and no clear plate drift was observed between plates tested. No effects of control treatment on calpain-2 and/or RPLP0 expression were observed when compared to vehicle treatment (fold changes and/or Cp values). Concordance between biological replicates was observed.
  • Oligonucleotide treatment Oligonucleotide compositions 14, 39, and TUG1 (negative control). Concentration range: 20 ⁇ M, 6.329 ⁇ M, 2.003 ⁇ M, 0.634 ⁇ M, 0.201 ⁇ M, 0.063 ⁇ M, 0.020 ⁇ M, 0.006 ⁇ M.
  • Positive control targeting NEAT1: oligonucleotide targeting NEAT1 (20 ⁇ M).
  • Vehicle 2% (v/v) TE buffer without oligonucleotides. Oligonucleotides were added to cells on day 7 after seeding the cells and refreshed on day 9 (by gymnotic uptake, 72 hours total incubation).
  • Cells can be harvested using Cells-to-Ct 1-Step TaqMan kit (Life Technologies), according to supplier’s instructions (cell lysis without separate RNA purification and one-step reverse transcription + real time PCR (real time RT-PCR). 25 ⁇ L/well lysis buffer. 3 days after treatment. [00196] RT-qPCR: 15% of Cells-to-Ct lysate samples (or water for non-template control; NTC). Single-plex reactions, in 384-well format, in technical duplicates. TaqMan Gene Expression Assay using LightCycler 480 equipment.
  • Oligonucleotide 14 can provide about 96% knock-down of CAPN2 at 20 uM at 72 hr. No significant effects on expression of RPLP0 were observed. The negative control oligonucleotide targeting TUG1 did not show a concentration-dependent effect on CAPN2 mRNA levels. Nonlinear regression (variable Hill slope) was used to convert EC50 values to pEC50 values. Example 5.
  • Various oligonucleotides and compositions can provide neuroprotective effects. [00200] Among other things, the present disclosure demonstrate that various provided oligonucleotide and compositions can provide neuroprotective effects, e.g., reduced Neurofilament-L (NF-L) excretion following exposure to toxic triggers.
  • NF-L Neurofilament-L
  • Cells iCell Motor Neurons, seeded at 32,000 cells/well in 96-well format. Thawing, seeding and culturing can be performed according to supplier’s instructions (e.g., coating: 0.07% PEI + 10 ⁇ g/ml Cultrex 3D mouse laminin; medium: complete maintenance medium with 20 mM DAPT for day 0 through day 6, complete maintenance medium without DAPT for day 7 through day 10).
  • Oligonucleotide treatment Oligonucleotide composition 14 and TUG1 (negative control). Concentration: 20 ⁇ M.
  • Negative control ⁇ A*C*C*A*G*>T*G*C*A*T*T*C*A*T*T* ⁇ C*G*A*G*T> (targeting TUG1); all nucleosides are DNA nucleosides except those in ⁇ > which are 2’-MOE modified, and each * independently represents a phosphorothioate internucleotidic linkage.
  • Toxicity triggers Various toxicity triggers were added on day 9.
  • Toxicity triggers used include vincristine (1.5 nM, 3 nM, 6 nM), rotenone (5 ⁇ M, 10 ⁇ M, 15 ⁇ M), and colchicine (10 nM, 100 nM, 1000 nM).
  • Neurofilament-L assay Supernatant from cells was harvested on day 10 at 24 hours after addition of toxicity triggers. Concentration of Neurofilament-L (NF-L) was determined using R-PLEX Human Neurofilament-L Assay kit (Meso Scale Discovery) according to supplier instructions.
  • pre-treatment with oligonucleotide composition 14 reduced toxicity trigger-induced NF-L excretion as compared to vehicle.
  • NF-L excretion following exposure to vincristine at 1.5 nM, 3 nM, or 6 nM; Figure 5, (A)), rotenone (at 5 ⁇ M, 10 ⁇ M, or 15 ⁇ M; Figure 5, (B)), or colchicine (100 nM; Figure 5, (C)) as compared to vehicle was observed with pre-treatment with oligonucleotide composition 14.
  • the negative control (targeting TUG1) oligonucleotide composition also reduced toxicity trigger-induced NF-L excretion as compared to vehicle. Without the intention to be limited by any theory, the observed reduction with the TUG1-targeted oligonucleotide composition may be due to a calpain-independent effect.
  • oligonucleotide composition 14 As compared to the TUG1-targeted oligonucleotide composition in various cases.
  • NF-L analysis by MSD showed an observable assay window of oligonucleotide composition 14 over TUG1.
  • oligonucleotide composition 14 When assessed in a neuritic degeneration assay, it appeared that oligonucleotide composition 14 partially prevented trigger induced neuritic degeneration, e.g., as measured by tubulin immunoreactivity area following exposure to toxicity triggers. In some embodiments, certain effects were also observed for TUG1-targeted oligonucleotide composition.
  • Example 6
  • Provided technologies can reduce reduction of calpain-2 mRNA and protein levels.
  • the present disclosure demonstrates that various provided oligonucleotide and compositions can provide reduction of calpain-2 mRNA and protein levels, in some instances, for days, weeks or longer. Results from certain assessments are presented below as an example. Those skilled in the art appreciate that other technologies can also be utilized to assess provided technologies in accordance with the present disclosure to confirm technical effects, benefits, advantages, etc. of provided technologies.
  • Cells iCell Motor Neurons, seeded at 32,000 cells/well in 96-well format. Thawing, seeding and culturing can be performed according to supplier’s instructions.
  • Oligonucleotide treatment Oligonucleotide compositions 14, 39, and TUG1 (negative control). Concentration: 20 ⁇ M for oligonucleotide composition 14, 18.9 ⁇ M for oligonucleotide composition 39, and 20 ⁇ M for negative control oligonucleotide targeting TUG1. Vehicle: 2% (v/v) TE buffer without oligonucleotides. Oligonucleotides were added to cells on day 7 after seeding the cells (by gymnotic uptake) and removed by medium refreshment on day 9 (48 hours total incubation).
  • Cells were collected and lysed on day 7 after seeding the cells (baseline) and 3, 7, 10, 14, and 21 days after removal of oligonucleotide compositions.
  • treatments were conducted in triplicate for each condition, with a separate plate for each collection time point.
  • treatments were conducted in three triplicates for each condition with a separate plate for each collection time point.
  • Negative control ⁇ A*C*C*A*G*>T*G*C*A*T*T*C*A*T*T* ⁇ C*G*A*G*T>; all nucleosides are DNA nucleosides except those in ⁇ > which are 2’-MOE modified, and each * independently represents a phosphorothioate internucleotidic linkage.
  • mRNA Harvesting Cells were harvested using Cells-to-Ct 1-Step TaqMan kit (Thermo Fisher), according to supplier’s instructions (25 ⁇ L/well lysis buffer) on day 7 after seeding the cells (baseline) and at 3, 7, 10, 14, and 21 days after removal of oligonucleotide compositions.
  • RT-PCR 15% of Cells-to-Ct lysate samples and the primer-probe sets in the table below, as single-plex reactions, in 384-well format, in technical duplicates. TaqMan Gene Expression Assay using LightCycler 480 equipment (Roche).
  • calpain-2 (CAPN2) mRNA knockdown was confirmed.
  • Oligonucleotide 14 can provide about 94% knockdown of calpain-2 mRNA at 20 ⁇ M on day 0 post-removal of oligonucleotide and about 77% knockdown of calpain-2 mRNA at 20 ⁇ M on day 21 post- removal of oligonucleotide.
  • Oligonucleotide 39 can provide about 87% knockdown of calpain-2 mRNA at 18.9 ⁇ M on day 0 post-removal of oligonucleotide and about 82% knockdown of calpain-2 mRNA at 18.9 ⁇ M on day 21 post-removal of oligonucleotide. Both oligonucleotide 14 and oligonucleotide 39 demonstrated persistent knockdown of calpain-2 mRNA from day 0 post-removal of oligonucleotide to at least day 21 post-removal of oligonucleotide. The negative control oligonucleotide targeting TUG1 did not display knockdown of calpain-2 mRNA levels.
  • Protein Harvesting Cell lysates prepared using RIPA lysis buffer (30 ⁇ L/well), including Halt protease inhibitor cocktail (Thermo Fisher). Three wells per replicate were pooled to ensure obtainment of sufficient amounts for protein detection. Three replicates were harvested per condition per time point. Protein yield was assessed by BCA assay.
  • Protein Detection Calpain-2 protein was detected using western blot via Jess equipment (ProteinSimple) according to supplier’s instructions. Two replicates for each condition and time point were examined.
  • Calpain-2 antibody (EPR5977/ab126600; Abcam) was used at a 1:6 dilution.
  • Protein Data Analysis Chemiluminescence-based detection of target immunoreactivity. Signal analysis for area under curve (AUC), detected molecular (MW), and signal-to-noise ratios using Compass software. Quantification of % target protein knockdown, normalized for total protein.
  • Calpain-2 protein levels were not affected by vehicle (TE buffer) or negative control treatment as compared to untreated cells at 48 hours after treatment. Some variation in calpain-2 protein levels between replicates was observed with some conditions at some time points, e.g., in some cases likely due to well-to-well variability in cell viability.
  • Oligonucleotide 14 can provide about 51% knockdown of calpain-2 protein at 20 ⁇ M at day 14 post- removal of oligonucleotide, as shown in Figure 7A.
  • Oligonucleotide 39 can provide about 60% knockdown of calpain-2 protein at 18.9 ⁇ M at day 14 post-removal of oligonucleotide, as shown in Figure 7B. Both oligonucleotide 14 and oligonucleotide 39 demonstrated persistent calpain-2 protein knockdown from day 7 post-removal of oligonucleotide and maintained persistent calpain-2 protein knockdown to at least day 21 post-removal of oligonucleotide.
  • the negative control oligonucleotide targeting TUG1 did not display knockdown of calpain-2 protein levels.
  • data of the present Example confirm that provided technologies can provide persistent knockdown of calpain-2 protein, e.g., following removal or washout of the oligonucleotide or composition.
  • Various other technologies are available for assessing properties and/or activities of provided technologies in accordance with the present disclosure. For example, reduction of levels of calpain-2 polypeptides can be assessed through western blot or immunostaining. In some embodiments, provided oligonucleotides and compositions are assessed for reduction of calpain-2 activation.
  • provided oligonucleotides and compositions are assessed for reduction of calpain-2 protease activity. In some embodiments, provided oligonucleotides and compositions are assessed for reduction of neuronal degeneration, e.g., through using one or more cell models. In some embodiments, provided oligonucleotides and compositions are assessed for enhanced recovery from axotomy. [00222] While various embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described in the present disclosure, and each of such variations and/or modifications is deemed to be included.

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Abstract

Entre autres, la présente invention concerne des oligonucléotides ciblant la calpaïne-2. modifiés à la position 2` du résidu sucre avec un groupe méthoxy, méthoxyéthoxy et des compositions de ceux-ci. Dans certains modes de réalisation, la présente invention concerne des procédés de prévention ou de traitement de divers états, troubles ou maladies, y compris d'états neurodégénératifs.
PCT/US2023/021586 2022-05-10 2023-05-09 Compositions d'oligonucléotides et procédés associés WO2023220087A1 (fr)

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